Validation of commercial ERK antibodies against the ERK orthologue of the scleractinian coral Stylophora pistillata

Validation of commercial ERK antibodies against the... | F1000Research "use strict";function _typeof(t){return(_typeof="function"==typeof Symbol&&"symbol"==typeof Symbol.iterator?function(t){return typeof t}:function(t){return t&&"function"==typeof Symbol&&t.constructor===Symbol&&t!==Symbol.prototype?"symbol":typeof t})(t)}!function(){var t=function(){var t,e,o=[],n=window,r=n;for(;r;){try{if(r.frames.__tcfapiLocator){t=r;break}}catch(t){}if(r===n.top)break;r=r.parent}t||(!function t(){var e=n.document,o=!!n.frames.__tcfapiLocator;if(!o)if(e.body){var r=e.createElement("iframe");r.style.cssText="display:none",r.name="__tcfapiLocator",e.body.appendChild(r)}else setTimeout(t,5);return!o}(),n.__tcfapi=function(){for(var t=arguments.length,n=new Array(t),r=0;r 3&&2===parseInt(n[1],10)&&"boolean"==typeof n[3]&&(e=n[3],"function"==typeof n[2]&&n[2]("set",!0)):"ping"===n[0]?"function"==typeof n[2]&&n[2]({gdprApplies:e,cmpLoaded:!1,cmpStatus:"stub"}):o.push(n)},n.addEventListener("message",(function(t){var e="string"==typeof t.data,o={};if(e)try{o=JSON.parse(t.data)}catch(t){}else o=t.data;var n="object"===_typeof(o)&&null!==o?o.__tcfapiCall:null;n&&window.__tcfapi(n.command,n.version,(function(o,r){var a={__tcfapiReturn:{returnValue:o,success:r,callId:n.callId}};t&&t.source&&t.source.postMessage&&t.source.postMessage(e?JSON.stringify(a):a,"*")}),n.parameter)}),!1))};"undefined"!=typeof module?module.exports=t:t()}(); dataLayer = dataLayer || []; // Standard GTM initialization - Google Consent Mode handles consent automatically (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start': new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0], j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src= 'https://www.googletagmanager.com/gtm.js?id='+i+dl+ '>m_auth=hzk0Vc3qFsQYhCrIoHz68A>m_preview=env-1>m_cookies_win=x';f.parentNode.insertBefore(j,f); })(window,document,'script','dataLayer','GTM-MWFK8L5J'); ;window.NREUM||(NREUM={});NREUM.init={distributed_tracing:{enabled:true},privacy:{cookies_enabled:true},ajax:{deny_list:["bam.nr-data.net"]}}; ;NREUM.loader_config={accountID:"438030",trustKey:"438030",agentID:"772317073",licenseKey:"97f8f67f26",applicationID:"772317073"} ;NREUM.info={beacon:"bam.nr-data.net",errorBeacon:"bam.nr-data.net",licenseKey:"97f8f67f26",applicationID:"772317073",sa:1} ;/*! For license information please see nr-loader-spa-1.236.0.min.js.LICENSE.txt */ (()=>{"use strict";var e,t,r={5763:(e,t,r)=>{r.d(t,{P_:()=>l,Mt:()=>g,C5:()=>s,DL:()=>v,OP:()=>T,lF:()=>D,Yu:()=>y,Dg:()=>h,CX:()=>c,GE:()=>b,sU:()=>_});var n=r(8632),i=r(9567);const o={beacon:n.ce.beacon,errorBeacon:n.ce.errorBeacon,licenseKey:void 0,applicationID:void 0,sa:void 0,queueTime:void 0,applicationTime:void 0,ttGuid:void 0,user:void 0,account:void 0,product:void 0,extra:void 0,jsAttributes:{},userAttributes:void 0,atts:void 0,transactionName:void 0,tNamePlain:void 0},a={};function s(e){if(!e)throw new Error("All info objects require an agent identifier!");if(!a[e])throw new Error("Info for ".concat(e," was never set"));return a[e]}function c(e,t){if(!e)throw new Error("All info objects require an agent identifier!");a[e]=(0,i.D)(t,o),(0,n.Qy)(e,a[e],"info")}var u=r(7056);const d=()=>{const e={blockSelector:"[data-nr-block]",maskInputOptions:{password:!0}};return{allow_bfcache:!0,privacy:{cookies_enabled:!0},ajax:{deny_list:void 0,enabled:!0,harvestTimeSeconds:10},distributed_tracing:{enabled:void 0,exclude_newrelic_header:void 0,cors_use_newrelic_header:void 0,cors_use_tracecontext_headers:void 0,allowed_origins:void 0},session:{domain:void 0,expiresMs:u.oD,inactiveMs:u.Hb},ssl:void 0,obfuscate:void 0,jserrors:{enabled:!0,harvestTimeSeconds:10},metrics:{enabled:!0},page_action:{enabled:!0,harvestTimeSeconds:30},page_view_event:{enabled:!0},page_view_timing:{enabled:!0,harvestTimeSeconds:30,long_task:!1},session_trace:{enabled:!0,harvestTimeSeconds:10},harvest:{tooManyRequestsDelay:60},session_replay:{enabled:!1,harvestTimeSeconds:60,sampleRate:.1,errorSampleRate:.1,maskTextSelector:"*",maskAllInputs:!0,get blockClass(){return"nr-block"},get ignoreClass(){return"nr-ignore"},get maskTextClass(){return"nr-mask"},get blockSelector(){return e.blockSelector},set blockSelector(t){e.blockSelector+=",".concat(t)},get maskInputOptions(){return e.maskInputOptions},set maskInputOptions(t){e.maskInputOptions={...t,password:!0}}},spa:{enabled:!0,harvestTimeSeconds:10}}},f={};function l(e){if(!e)throw new Error("All configuration objects require an agent identifier!");if(!f[e])throw new Error("Configuration for ".concat(e," was never set"));return f[e]}function h(e,t){if(!e)throw new Error("All configuration objects require an agent identifier!");f[e]=(0,i.D)(t,d()),(0,n.Qy)(e,f[e],"config")}function g(e,t){if(!e)throw new Error("All configuration objects require an agent identifier!");var r=l(e);if(r){for(var n=t.split("."),i=0;i {r.d(t,{D:()=>i});var n=r(50);function i(e,t){try{if(!e||"object"!=typeof e)return(0,n.Z)("Setting a Configurable requires an object as input");if(!t||"object"!=typeof t)return(0,n.Z)("Setting a Configurable requires a model to set its initial properties");const r=Object.create(Object.getPrototypeOf(t),Object.getOwnPropertyDescriptors(t)),o=0===Object.keys(r).length?e:r;for(let a in o)if(void 0!==e[a])try{"object"==typeof e[a]&&"object"==typeof t[a]?r[a]=i(e[a],t[a]):r[a]=e[a]}catch(e){(0,n.Z)("An error occurred while setting a property of a Configurable",e)}return r}catch(e){(0,n.Z)("An error occured while setting a Configurable",e)}}},6818:(e,t,r)=>{r.d(t,{Re:()=>i,gF:()=>o,q4:()=>n});const n="1.236.0",i="PROD",o="CDN"},385:(e,t,r)=>{r.d(t,{FN:()=>a,IF:()=>u,Nk:()=>f,Tt:()=>s,_A:()=>o,il:()=>n,pL:()=>c,v6:()=>i,w1:()=>d});const n="undefined"!=typeof window&&!!window.document,i="undefined"!=typeof WorkerGlobalScope&&("undefined"!=typeof self&&self instanceof WorkerGlobalScope&&self.navigator instanceof WorkerNavigator||"undefined"!=typeof globalThis&&globalThis instanceof WorkerGlobalScope&&globalThis.navigator instanceof WorkerNavigator),o=n?window:"undefined"!=typeof WorkerGlobalScope&&("undefined"!=typeof self&&self instanceof WorkerGlobalScope&&self||"undefined"!=typeof globalThis&&globalThis instanceof WorkerGlobalScope&&globalThis),a=""+o?.location,s=/iPad|iPhone|iPod/.test(navigator.userAgent),c=s&&"undefined"==typeof SharedWorker,u=(()=>{const e=navigator.userAgent.match(/Firefox[/\s](\d+\.\d+)/);return Array.isArray(e)&&e.length>=2?+e[1]:0})(),d=Boolean(n&&window.document.documentMode),f=!!navigator.sendBeacon},1117:(e,t,r)=>{r.d(t,{w:()=>o});var n=r(50);const i={agentIdentifier:"",ee:void 0};class o{constructor(e){try{if("object"!=typeof e)return(0,n.Z)("shared context requires an object as input");this.sharedContext={},Object.assign(this.sharedContext,i),Object.entries(e).forEach((e=>{let[t,r]=e;Object.keys(i).includes(t)&&(this.sharedContext[t]=r)}))}catch(e){(0,n.Z)("An error occured while setting SharedContext",e)}}}},8e3:(e,t,r)=>{r.d(t,{L:()=>d,R:()=>c});var n=r(2177),i=r(1284),o=r(4322),a=r(3325);const s={};function c(e,t){const r={staged:!1,priority:a.p[t]||0};u(e),s[e].get(t)||s[e].set(t,r)}function u(e){e&&(s[e]||(s[e]=new Map))}function d(){let e=arguments.length>0&&void 0!==arguments[0]?arguments[0]:"",t=arguments.length>1&&void 0!==arguments[1]?arguments[1]:"feature";if(u(e),!e||!s[e].get(t))return a(t);s[e].get(t).staged=!0;const r=[...s[e]];function a(t){const r=e?n.ee.get(e):n.ee,a=o.X.handlers;if(r.backlog&&a){var s=r.backlog[t],c=a[t];if(c){for(var u=0;s&&u {let[t,r]=e;return r.staged}))&&(r.sort(((e,t)=>e[1].priority-t[1].priority)),r.forEach((e=>{let[t]=e;a(t)})))}function f(e,t){var r=e[1];(0,i.D)(t[r],(function(t,r){var n=e[0];if(r[0]===n){var i=r[1],o=e[3],a=e[2];i.apply(o,a)}}))}},2177:(e,t,r)=>{r.d(t,{c:()=>f,ee:()=>u});var n=r(8632),i=r(2210),o=r(1284),a=r(5763),s="nr@context";let c=(0,n.fP)();var u;function d(){}function f(e){return(0,i.X)(e,s,l)}function l(){return new d}function h(){u.aborted=!0,u.backlog={}}c.ee?u=c.ee:(u=function e(t,r){var n={},c={},f={},g=!1;try{g=16===r.length&&(0,a.OP)(r).isolatedBacklog}catch(e){}var p={on:b,addEventListener:b,removeEventListener:y,emit:v,get:x,listeners:w,context:m,buffer:A,abort:h,aborted:!1,isBuffering:E,debugId:r,backlog:g?{}:t&&"object"==typeof t.backlog?t.backlog:{}};return p;function m(e){return e&&e instanceof d?e:e?(0,i.X)(e,s,l):l()}function v(e,r,n,i,o){if(!1!==o&&(o=!0),!u.aborted||i){t&&o&&t.emit(e,r,n);for(var a=m(n),s=w(e),d=s.length,f=0;fn,p:()=>i});var n=r(2177).ee.get("handle");function i(e,t,r,i,o){o?(o.buffer([e],i),o.emit(e,t,r)):(n.buffer([e],i),n.emit(e,t,r))}},4322:(e,t,r)=>{r.d(t,{X:()=>o});var n=r(5546);o.on=a;var i=o.handlers={};function o(e,t,r,o){a(o||n.E,i,e,t,r)}function a(e,t,r,i,o){o||(o="feature"),e||(e=n.E);var a=t[o]=t[o]||{};(a[r]=a[r]||[]).push([e,i])}},3239:(e,t,r)=>{r.d(t,{bP:()=>s,iz:()=>c,m$:()=>a});var n=r(385);let i=!1,o=!1;try{const e={get passive(){return i=!0,!1},get signal(){return o=!0,!1}};n._A.addEventListener("test",null,e),n._A.removeEventListener("test",null,e)}catch(e){}function a(e,t){return i||o?{capture:!!e,passive:i,signal:t}:!!e}function s(e,t){let r=arguments.length>2&&void 0!==arguments[2]&&arguments[2],n=arguments.length>3?arguments[3]:void 0;window.addEventListener(e,t,a(r,n))}function c(e,t){let r=arguments.length>2&&void 0!==arguments[2]&&arguments[2],n=arguments.length>3?arguments[3]:void 0;document.addEventListener(e,t,a(r,n))}},4402:(e,t,r)=>{r.d(t,{Ht:()=>u,M:()=>c,Rl:()=>a,ky:()=>s});var n=r(385);const i="xxxxxxxx-xxxx-4xxx-yxxx-xxxxxxxxxxxx";function o(e,t){return e?15&e[t]:16*Math.random()|0}function a(){const e=n._A?.crypto||n._A?.msCrypto;let t,r=0;return e&&e.getRandomValues&&(t=e.getRandomValues(new Uint8Array(31))),i.split("").map((e=>"x"===e?o(t,++r).toString(16):"y"===e?(3&o()|8).toString(16):e)).join("")}function s(e){const t=n._A?.crypto||n._A?.msCrypto;let r,i=0;t&&t.getRandomValues&&(r=t.getRandomValues(new Uint8Array(31)));const a=[];for(var s=0;s {r.d(t,{Bq:()=>n,Hb:()=>o,oD:()=>i});const n="NRBA",i=144e5,o=18e5},7894:(e,t,r)=>{function n(){return Math.round(performance.now())}r.d(t,{z:()=>n})},7243:(e,t,r)=>{r.d(t,{e:()=>o});var n=r(385),i={};function o(e){if(e in i)return i[e];if(0===(e||"").indexOf("data:"))return{protocol:"data"};let t;var r=n._A?.location,o={};if(n.il)t=document.createElement("a"),t.href=e;else try{t=new URL(e,r.href)}catch(e){return o}o.port=t.port;var a=t.href.split("://");!o.port&&a[1]&&(o.port=a[1].split("/")[0].split("@").pop().split(":")[1]),o.port&&"0"!==o.port||(o.port="https"===a[0]?"443":"80"),o.hostname=t.hostname||r.hostname,o.pathname=t.pathname,o.protocol=a[0],"/"!==o.pathname.charAt(0)&&(o.pathname="/"+o.pathname);var s=!t.protocol||":"===t.protocol||t.protocol===r.protocol,c=t.hostname===r.hostname&&t.port===r.port;return o.sameOrigin=s&&(!t.hostname||c),"/"===o.pathname&&(i[e]=o),o}},50:(e,t,r)=>{function n(e,t){"function"==typeof console.warn&&(console.warn("New Relic: ".concat(e)),t&&console.warn(t))}r.d(t,{Z:()=>n})},2587:(e,t,r)=>{r.d(t,{N:()=>c,T:()=>u});var n=r(2177),i=r(5546),o=r(8e3),a=r(3325);const s={stn:[a.D.sessionTrace],err:[a.D.jserrors,a.D.metrics],ins:[a.D.pageAction],spa:[a.D.spa],sr:[a.D.sessionReplay,a.D.sessionTrace]};function c(e,t){const r=n.ee.get(t);e&&"object"==typeof e&&(Object.entries(e).forEach((e=>{let[t,n]=e;void 0===u[t]&&(s[t]?s[t].forEach((e=>{n?(0,i.p)("feat-"+t,[],void 0,e,r):(0,i.p)("block-"+t,[],void 0,e,r),(0,i.p)("rumresp-"+t,[Boolean(n)],void 0,e,r)})):n&&(0,i.p)("feat-"+t,[],void 0,void 0,r),u[t]=Boolean(n))})),Object.keys(s).forEach((e=>{void 0===u[e]&&(s[e]?.forEach((t=>(0,i.p)("rumresp-"+e,[!1],void 0,t,r))),u[e]=!1)})),(0,o.L)(t,a.D.pageViewEvent))}const u={}},2210:(e,t,r)=>{r.d(t,{X:()=>i});var n=Object.prototype.hasOwnProperty;function i(e,t,r){if(n.call(e,t))return e[t];var i=r();if(Object.defineProperty&&Object.keys)try{return Object.defineProperty(e,t,{value:i,writable:!0,enumerable:!1}),i}catch(e){}return e[t]=i,i}},1284:(e,t,r)=>{r.d(t,{D:()=>n});const n=(e,t)=>Object.entries(e||{}).map((e=>{let[r,n]=e;return t(r,n)}))},4351:(e,t,r)=>{r.d(t,{P:()=>o});var n=r(2177);const i=()=>{const e=new WeakSet;return(t,r)=>{if("object"==typeof r&&null!==r){if(e.has(r))return;e.add(r)}return r}};function o(e){try{return JSON.stringify(e,i())}catch(e){try{n.ee.emit("internal-error",[e])}catch(e){}}}},3960:(e,t,r)=>{r.d(t,{K:()=>a,b:()=>o});var n=r(3239);function i(){return"undefined"==typeof document||"complete"===document.readyState}function o(e,t){if(i())return e();(0,n.bP)("load",e,t)}function a(e){if(i())return e();(0,n.iz)("DOMContentLoaded",e)}},8632:(e,t,r)=>{r.d(t,{EZ:()=>u,Qy:()=>c,ce:()=>o,fP:()=>a,gG:()=>d,mF:()=>s});var n=r(7894),i=r(385);const o={beacon:"bam.nr-data.net",errorBeacon:"bam.nr-data.net"};function a(){return i._A.NREUM||(i._A.NREUM={}),void 0===i._A.newrelic&&(i._A.newrelic=i._A.NREUM),i._A.NREUM}function s(){let e=a();return e.o||(e.o={ST:i._A.setTimeout,SI:i._A.setImmediate,CT:i._A.clearTimeout,XHR:i._A.XMLHttpRequest,REQ:i._A.Request,EV:i._A.Event,PR:i._A.Promise,MO:i._A.MutationObserver,FETCH:i._A.fetch}),e}function c(e,t,r){let i=a();const o=i.initializedAgents||{},s=o[e]||{};return Object.keys(s).length||(s.initializedAt={ms:(0,n.z)(),date:new Date}),i.initializedAgents={...o,[e]:{...s,[r]:t}},i}function u(e,t){a()[e]=t}function d(){return function(){let e=a();const t=e.info||{};e.info={beacon:o.beacon,errorBeacon:o.errorBeacon,...t}}(),function(){let e=a();const t=e.init||{};e.init={...t}}(),s(),function(){let e=a();const t=e.loader_config||{};e.loader_config={...t}}(),a()}},7956:(e,t,r)=>{r.d(t,{N:()=>i});var n=r(3239);function i(e){let t=arguments.length>1&&void 0!==arguments[1]&&arguments[1],r=arguments.length>2?arguments[2]:void 0,i=arguments.length>3?arguments[3]:void 0;return void(0,n.iz)("visibilitychange",(function(){if(t)return void("hidden"==document.visibilityState&&e());e(document.visibilityState)}),r,i)}},1214:(e,t,r)=>{r.d(t,{em:()=>v,u5:()=>N,QU:()=>S,_L:()=>I,Gm:()=>L,Lg:()=>M,gy:()=>U,BV:()=>Q,Kf:()=>ee});var n=r(2177);const i="nr@original";var o=Object.prototype.hasOwnProperty,a=!1;function s(e,t){return e||(e=n.ee),r.inPlace=function(e,t,n,i,o){n||(n="");var a,s,c,u="-"===n.charAt(0);for(c=0;c 2?n-2:0),o=2;o {r(A[T],e,w),r(E[T],e,w)})),r(l._A,"fetch",y),t.on(y+"end",(function(e,r){var n=this;if(r){var i=r.headers.get("content-length");null!==i&&(n.rxSize=i),t.emit(y+"done",[null,r],n)}else t.emit(y+"done",[e],n)})),t}const O={},j=["pushState","replaceState"];function S(e){const t=function(e){return(e||n.ee).get("history")}(e);return!l.il||O[t.debugId]++||(O[t.debugId]=1,s(t).inPlace(window.history,j,"-")),t}var P=r(3239);const C={},R=["appendChild","insertBefore","replaceChild"];function I(e){const t=function(e){return(e||n.ee).get("jsonp")}(e);if(!l.il||C[t.debugId])return t;C[t.debugId]=!0;var r=s(t),i=/[?&](?:callback|cb)=([^&#]+)/,o=/(.*)\.([^.]+)/,a=/^(\w+)(\.|$)(.*)$/;function c(e,t){var r=e.match(a),n=r[1],i=r[3];return i?c(i,t[n]):t[n]}return r.inPlace(Node.prototype,R,"dom-"),t.on("dom-start",(function(e){!function(e){if(!e||"string"!=typeof e.nodeName||"script"!==e.nodeName.toLowerCase())return;if("function"!=typeof e.addEventListener)return;var n=(a=e.src,s=a.match(i),s?s[1]:null);var a,s;if(!n)return;var u=function(e){var t=e.match(o);if(t&&t.length>=3)return{key:t[2],parent:c(t[1],window)};return{key:e,parent:window}}(n);if("function"!=typeof u.parent[u.key])return;var d={};function f(){t.emit("jsonp-end",[],d),e.removeEventListener("load",f,(0,P.m$)(!1)),e.removeEventListener("error",l,(0,P.m$)(!1))}function l(){t.emit("jsonp-error",[],d),t.emit("jsonp-end",[],d),e.removeEventListener("load",f,(0,P.m$)(!1)),e.removeEventListener("error",l,(0,P.m$)(!1))}r.inPlace(u.parent,[u.key],"cb-",d),e.addEventListener("load",f,(0,P.m$)(!1)),e.addEventListener("error",l,(0,P.m$)(!1)),t.emit("new-jsonp",[e.src],d)}(e[0])})),t}var k=r(5763);const H={};function L(e){const t=function(e){return(e||n.ee).get("mutation")}(e);if(!l.il||H[t.debugId])return t;H[t.debugId]=!0;var r=s(t),i=k.Yu.MO;return i&&(window.MutationObserver=function(e){return this instanceof i?new i(r(e,"fn-")):i.apply(this,arguments)},MutationObserver.prototype=i.prototype),t}const z={};function M(e){const t=function(e){return(e||n.ee).get("promise")}(e);if(z[t.debugId])return t;z[t.debugId]=!0;var r=n.c,o=s(t),a=k.Yu.PR;return a&&function(){function e(r){var n=t.context(),i=o(r,"executor-",n,null,!1);const s=Reflect.construct(a,[i],e);return t.context(s).getCtx=function(){return n},s}l._A.Promise=e,Object.defineProperty(e,"name",{value:"Promise"}),e.toString=function(){return a.toString()},Object.setPrototypeOf(e,a),["all","race"].forEach((function(r){const n=a[r];e[r]=function(e){let i=!1;[...e||[]].forEach((e=>{this.resolve(e).then(a("all"===r),a(!1))}));const o=n.apply(this,arguments);return o;function a(e){return function(){t.emit("propagate",[null,!i],o,!1,!1),i=i||!e}}}})),["resolve","reject"].forEach((function(r){const n=a[r];e[r]=function(e){const r=n.apply(this,arguments);return e!==r&&t.emit("propagate",[e,!0],r,!1,!1),r}})),e.prototype=a.prototype;const n=a.prototype.then;a.prototype.then=function(){var e=this,i=r(e);i.promise=e;for(var a=arguments.length,s=new Array(a),c=0;c e())),t};function m(e,t){i.inPlace(t,["onreadystatechange"],"fn-",E)}function b(){var e=this,t=r.context(e);e.readyState>3&&!t.resolved&&(t.resolved=!0,r.emit("xhr-resolved",[],e)),i.inPlace(e,f,"fn-",E)}if(function(e,t){for(var r in e)t[r]=e[r]}(o,p),p.prototype=o.prototype,i.inPlace(p.prototype,J,"-xhr-",E),r.on("send-xhr-start",(function(e,t){m(e,t),function(e){h.push(e),a&&(y?y.then(A):u?u(A):(w=-w,x.data=w))}(t)})),r.on("open-xhr-start",m),a){var y=c&&c.resolve();if(!u&&!c){var w=1,x=document.createTextNode(w);new a(A).observe(x,{characterData:!0})}}else t.on("fn-end",(function(e){e[0]&&e[0].type===d||A()}));function A(){for(var e=0;e {r.d(t,{t:()=>n});const n=r(3325).D.ajax},6660:(e,t,r)=>{r.d(t,{A:()=>i,t:()=>n});const n=r(3325).D.jserrors,i="nr@seenError"},3081:(e,t,r)=>{r.d(t,{gF:()=>o,mY:()=>i,t9:()=>n,vz:()=>s,xS:()=>a});const n=r(3325).D.metrics,i="sm",o="cm",a="storeSupportabilityMetrics",s="storeEventMetrics"},4649:(e,t,r)=>{r.d(t,{t:()=>n});const n=r(3325).D.pageAction},7633:(e,t,r)=>{r.d(t,{Dz:()=>i,OJ:()=>a,qw:()=>o,t9:()=>n});const n=r(3325).D.pageViewEvent,i="firstbyte",o="domcontent",a="windowload"},9251:(e,t,r)=>{r.d(t,{t:()=>n});const n=r(3325).D.pageViewTiming},3614:(e,t,r)=>{r.d(t,{BST_RESOURCE:()=>i,END:()=>s,FEATURE_NAME:()=>n,FN_END:()=>u,FN_START:()=>c,PUSH_STATE:()=>d,RESOURCE:()=>o,START:()=>a});const n=r(3325).D.sessionTrace,i="bstResource",o="resource",a="-start",s="-end",c="fn"+a,u="fn"+s,d="pushState"},7836:(e,t,r)=>{r.d(t,{BODY:()=>A,CB_END:()=>E,CB_START:()=>u,END:()=>x,FEATURE_NAME:()=>i,FETCH:()=>_,FETCH_BODY:()=>v,FETCH_DONE:()=>m,FETCH_START:()=>p,FN_END:()=>c,FN_START:()=>s,INTERACTION:()=>l,INTERACTION_API:()=>d,INTERACTION_EVENTS:()=>o,JSONP_END:()=>b,JSONP_NODE:()=>g,JS_TIME:()=>T,MAX_TIMER_BUDGET:()=>a,REMAINING:()=>f,SPA_NODE:()=>h,START:()=>w,originalSetTimeout:()=>y});var n=r(5763);const i=r(3325).D.spa,o=["click","submit","keypress","keydown","keyup","change"],a=999,s="fn-start",c="fn-end",u="cb-start",d="api-ixn-",f="remaining",l="interaction",h="spaNode",g="jsonpNode",p="fetch-start",m="fetch-done",v="fetch-body-",b="jsonp-end",y=n.Yu.ST,w="-start",x="-end",A="-body",E="cb"+x,T="jsTime",_="fetch"},5938:(e,t,r)=>{r.d(t,{W:()=>o});var n=r(5763),i=r(2177);class o{constructor(e,t,r){this.agentIdentifier=e,this.aggregator=t,this.ee=i.ee.get(e,(0,n.OP)(this.agentIdentifier).isolatedBacklog),this.featureName=r,this.blocked=!1}}},9144:(e,t,r)=>{r.d(t,{j:()=>m});var n=r(3325),i=r(5763),o=r(5546),a=r(2177),s=r(7894),c=r(8e3),u=r(3960),d=r(385),f=r(50),l=r(3081),h=r(8632);function g(){const e=(0,h.gG)();["setErrorHandler","finished","addToTrace","inlineHit","addRelease","addPageAction","setCurrentRouteName","setPageViewName","setCustomAttribute","interaction","noticeError","setUserId"].forEach((t=>{e[t]=function(){for(var r=arguments.length,n=new Array(r),i=0;i 1?r-1:0),i=1;i {e.exposed&&e.api[t]&&o.push(e.api[t](...n))})),o.length>1?o:o[0]}(t,...n)}}))}var p=r(2587);function m(e){let t=arguments.length>1&&void 0!==arguments[1]?arguments[1]:{},m=arguments.length>2?arguments[2]:void 0,v=arguments.length>3?arguments[3]:void 0,{init:b,info:y,loader_config:w,runtime:x={loaderType:m},exposed:A=!0}=t;const E=(0,h.gG)();y||(b=E.init,y=E.info,w=E.loader_config),(0,i.Dg)(e,b||{}),(0,i.GE)(e,w||{}),(0,i.sU)(e,x),y.jsAttributes??={},d.v6&&(y.jsAttributes.isWorker=!0),(0,i.CX)(e,y),g();const T=function(e,t){t||(0,c.R)(e,"api");const h={};var g=a.ee.get(e),p=g.get("tracer"),m="api-",v=m+"ixn-";function b(t,r,n,o){const a=(0,i.C5)(e);return null===r?delete a.jsAttributes[t]:(0,i.CX)(e,{...a,jsAttributes:{...a.jsAttributes,[t]:r}}),x(m,n,!0,o||null===r?"session":void 0)(t,r)}function y(){}["setErrorHandler","finished","addToTrace","inlineHit","addRelease"].forEach((e=>h[e]=x(m,e,!0,"api"))),h.addPageAction=x(m,"addPageAction",!0,n.D.pageAction),h.setCurrentRouteName=x(m,"routeName",!0,n.D.spa),h.setPageViewName=function(t,r){if("string"==typeof t)return"/"!==t.charAt(0)&&(t="/"+t),(0,i.OP)(e).customTransaction=(r||"http://custom.transaction")+t,x(m,"setPageViewName",!0)()},h.setCustomAttribute=function(e,t){let r=arguments.length>2&&void 0!==arguments[2]&&arguments[2];if("string"==typeof e){if(["string","number"].includes(typeof t)||null===t)return b(e,t,"setCustomAttribute",r);(0,f.Z)("Failed to execute setCustomAttribute.\nNon-null value must be a string or number type, but a type of was provided."))}else(0,f.Z)("Failed to execute setCustomAttribute.\nName must be a string type, but a type of was provided."))},h.setUserId=function(e){if("string"==typeof e||null===e)return b("enduser.id",e,"setUserId",!0);(0,f.Z)("Failed to execute setUserId.\nNon-null value must be a string type, but a type of was provided."))},h.interaction=function(){return(new y).get()};var w=y.prototype={createTracer:function(e,t){var r={},i=this,a="function"==typeof t;return(0,o.p)(v+"tracer",[(0,s.z)(),e,r],i,n.D.spa,g),function(){if(p.emit((a?"":"no-")+"fn-start",[(0,s.z)(),i,a],r),a)try{return t.apply(this,arguments)}catch(e){throw p.emit("fn-err",[arguments,this,"string"==typeof e?new Error(e):e],r),e}finally{p.emit("fn-end",[(0,s.z)()],r)}}}};function x(e,t,r,i){return function(){return(0,o.p)(l.xS,["API/"+t+"/called"],void 0,n.D.metrics,g),i&&(0,o.p)(e+t,[(0,s.z)(),...arguments],r?null:this,i,g),r?void 0:this}}function A(){r.e(439).then(r.bind(r,7438)).then((t=>{let{setAPI:r}=t;r(e),(0,c.L)(e,"api")})).catch((()=>(0,f.Z)("Downloading runtime APIs failed...")))}return["actionText","setName","setAttribute","save","ignore","onEnd","getContext","end","get"].forEach((e=>{w[e]=x(v,e,void 0,n.D.spa)})),h.noticeError=function(e,t){"string"==typeof e&&(e=new Error(e)),(0,o.p)(l.xS,["API/noticeError/called"],void 0,n.D.metrics,g),(0,o.p)("err",[e,(0,s.z)(),!1,t],void 0,n.D.jserrors,g)},d.il?(0,u.b)((()=>A()),!0):A(),h}(e,v);return(0,h.Qy)(e,T,"api"),(0,h.Qy)(e,A,"exposed"),(0,h.EZ)("activatedFeatures",p.T),T}},3325:(e,t,r)=>{r.d(t,{D:()=>n,p:()=>i});const n={ajax:"ajax",jserrors:"jserrors",metrics:"metrics",pageAction:"page_action",pageViewEvent:"page_view_event",pageViewTiming:"page_view_timing",sessionReplay:"session_replay",sessionTrace:"session_trace",spa:"spa"},i={[n.pageViewEvent]:1,[n.pageViewTiming]:2,[n.metrics]:3,[n.jserrors]:4,[n.ajax]:5,[n.sessionTrace]:6,[n.pageAction]:7,[n.spa]:8,[n.sessionReplay]:9}}},n={};function i(e){var t=n[e];if(void 0!==t)return t.exports;var o=n[e]={exports:{}};return r[e](o,o.exports,i),o.exports}i.m=r,i.d=(e,t)=>{for(var r in t)i.o(t,r)&&!i.o(e,r)&&Object.defineProperty(e,r,{enumerable:!0,get:t[r]})},i.f={},i.e=e=>Promise.all(Object.keys(i.f).reduce(((t,r)=>(i.f[r](e,t),t)),[])),i.u=e=>(({78:"page_action-aggregate",147:"metrics-aggregate",242:"session-manager",317:"jserrors-aggregate",348:"page_view_timing-aggregate",412:"lazy-feature-loader",439:"async-api",538:"recorder",590:"session_replay-aggregate",675:"compressor",733:"session_trace-aggregate",786:"page_view_event-aggregate",873:"spa-aggregate",898:"ajax-aggregate"}[e]||e)+"."+{78:"ac76d497",147:"3dc53903",148:"1a20d5fe",242:"2a64278a",317:"49e41428",348:"bd6de33a",412:"2f55ce66",439:"30bd804e",538:"1b18459f",590:"cf0efb30",675:"ae9f91a8",733:"83105561",786:"06482edd",860:"03a8b7a5",873:"e6b09d52",898:"998ef92b"}[e]+"-1.236.0.min.js"),i.o=(e,t)=>Object.prototype.hasOwnProperty.call(e,t),e={},t="NRBA:",i.l=(r,n,o,a)=>{if(e[r])e[r].push(n);else{var s,c;if(void 0!==o)for(var u=document.getElementsByTagName("script"),d=0;d {s.onerror=s.onload=null,clearTimeout(h);var i=e[r];if(delete e[r],s.parentNode&&s.parentNode.removeChild(s),i&&i.forEach((e=>e(n))),t)return t(n)},h=setTimeout(l.bind(null,void 0,{type:"timeout",target:s}),12e4);s.onerror=l.bind(null,s.onerror),s.onload=l.bind(null,s.onload),c&&document.head.appendChild(s)}},i.r=e=>{"undefined"!=typeof Symbol&&Symbol.toStringTag&&Object.defineProperty(e,Symbol.toStringTag,{value:"Module"}),Object.defineProperty(e,"__esModule",{value:!0})},i.j=364,i.p="https://js-agent.newrelic.com/",(()=>{var e={364:0,953:0};i.f.j=(t,r)=>{var n=i.o(e,t)?e[t]:void 0;if(0!==n)if(n)r.push(n[2]);else{var o=new Promise(((r,i)=>n=e[t]=[r,i]));r.push(n[2]=o);var a=i.p+i.u(t),s=new Error;i.l(a,(r=>{if(i.o(e,t)&&(0!==(n=e[t])&&(e[t]=void 0),n)){var o=r&&("load"===r.type?"missing":r.type),a=r&&r.target&&r.target.src;s.message="Loading chunk "+t+" failed.\n("+o+": "+a+")",s.name="ChunkLoadError",s.type=o,s.request=a,n[1](s)}}),"chunk-"+t,t)}};var t=(t,r)=>{var n,o,[a,s,c]=r,u=0;if(a.some((t=>0!==e[t]))){for(n in s)i.o(s,n)&&(i.m[n]=s[n]);if(c)c(i)}for(t&&t(r);u {i.r(o);var e=i(3325),t=i(5763);const r=Object.values(e.D);function n(e){const n={};return r.forEach((r=>{n[r]=function(e,r){return!1!==(0,t.Mt)(r,"".concat(e,".enabled"))}(r,e)})),n}var a=i(9144);var s=i(5546),c=i(385),u=i(8e3),d=i(5938),f=i(3960),l=i(50);class h extends d.W{constructor(e,t,r){let n=!(arguments.length>3&&void 0!==arguments[3])||arguments[3];super(e,t,r),this.auto=n,this.abortHandler,this.featAggregate,this.onAggregateImported,n&&(0,u.R)(e,r)}importAggregator(){let e=arguments.length>0&&void 0!==arguments[0]?arguments[0]:{};if(this.featAggregate||!this.auto)return;const r=c.il&&!0===(0,t.Mt)(this.agentIdentifier,"privacy.cookies_enabled");let n;this.onAggregateImported=new Promise((e=>{n=e}));const o=async()=>{let t;try{if(r){const{setupAgentSession:e}=await Promise.all([i.e(860),i.e(242)]).then(i.bind(i,3228));t=e(this.agentIdentifier)}}catch(e){(0,l.Z)("A problem occurred when starting up session manager. This page will not start or extend any session.",e)}try{if(!this.shouldImportAgg(this.featureName,t))return void(0,u.L)(this.agentIdentifier,this.featureName);const{lazyFeatureLoader:r}=await i.e(412).then(i.bind(i,8582)),{Aggregate:o}=await r(this.featureName,"aggregate");this.featAggregate=new o(this.agentIdentifier,this.aggregator,e),n(!0)}catch(e){(0,l.Z)("Downloading and initializing ".concat(this.featureName," failed..."),e),this.abortHandler?.(),n(!1)}};c.il?(0,f.b)((()=>o()),!0):o()}shouldImportAgg(r,n){return r!==e.D.sessionReplay||!1!==(0,t.Mt)(this.agentIdentifier,"session_trace.enabled")&&(!!n?.isNew||!!n?.state.sessionReplay)}}var g=i(7633),p=i(7894);class m extends h{static featureName=g.t9;constructor(r,n){let i=!(arguments.length>2&&void 0!==arguments[2])||arguments[2];if(super(r,n,g.t9,i),("undefined"==typeof PerformanceNavigationTiming||c.Tt)&&"undefined"!=typeof PerformanceTiming){const n=(0,t.OP)(r);n[g.Dz]=Math.max(Date.now()-n.offset,0),(0,f.K)((()=>n[g.qw]=Math.max((0,p.z)()-n[g.Dz],0))),(0,f.b)((()=>{const t=(0,p.z)();n[g.OJ]=Math.max(t-n[g.Dz],0),(0,s.p)("timing",["load",t],void 0,e.D.pageViewTiming,this.ee)}))}this.importAggregator()}}var v=i(1117),b=i(1284);class y extends v.w{constructor(e){super(e),this.aggregatedData={}}store(e,t,r,n,i){var o=this.getBucket(e,t,r,i);return o.metrics=function(e,t){t||(t={count:0});return t.count+=1,(0,b.D)(e,(function(e,r){t[e]=w(r,t[e])})),t}(n,o.metrics),o}merge(e,t,r,n,i){var o=this.getBucket(e,t,n,i);if(o.metrics){var a=o.metrics;a.count+=r.count,(0,b.D)(r,(function(e,t){if("count"!==e){var n=a[e],i=r[e];i&&!i.c?a[e]=w(i.t,n):a[e]=function(e,t){if(!t)return e;t.c||(t=x(t.t));return t.min=Math.min(e.min,t.min),t.max=Math.max(e.max,t.max),t.t+=e.t,t.sos+=e.sos,t.c+=e.c,t}(i,a[e])}}))}else o.metrics=r}storeMetric(e,t,r,n){var i=this.getBucket(e,t,r);return i.stats=w(n,i.stats),i}getBucket(e,t,r,n){this.aggregatedData[e]||(this.aggregatedData[e]={});var i=this.aggregatedData[e][t];return i||(i=this.aggregatedData[e][t]={params:r||{}},n&&(i.custom=n)),i}get(e,t){return t?this.aggregatedData[e]&&this.aggregatedData[e][t]:this.aggregatedData[e]}take(e){for(var t={},r="",n=!1,i=0;i t.max&&(t.max=e),e 2&&void 0!==arguments[2])||arguments[2];super(e,r,j.t,n),c.il&&((0,t.OP)(e).initHidden=Boolean("hidden"===document.visibilityState),(0,N.N)((()=>(0,s.p)("docHidden",[(0,p.z)()],void 0,j.t,this.ee)),!0),(0,O.bP)("pagehide",(()=>(0,s.p)("winPagehide",[(0,p.z)()],void 0,j.t,this.ee))),this.importAggregator())}}var P=i(3081);class C extends h{static featureName=P.t9;constructor(e,t){let r=!(arguments.length>2&&void 0!==arguments[2])||arguments[2];super(e,t,P.t9,r),this.importAggregator()}}var R,I=i(2210),k=i(1214),H=i(2177),L={};try{R=localStorage.getItem("__nr_flags").split(","),console&&"function"==typeof console.log&&(L.console=!0,-1!==R.indexOf("dev")&&(L.dev=!0),-1!==R.indexOf("nr_dev")&&(L.nrDev=!0))}catch(e){}function z(e){try{L.console&&z(e)}catch(e){}}L.nrDev&&H.ee.on("internal-error",(function(e){z(e.stack)})),L.dev&&H.ee.on("fn-err",(function(e,t,r){z(r.stack)})),L.dev&&(z("NR AGENT IN DEVELOPMENT MODE"),z("flags: "+(0,b.D)(L,(function(e,t){return e})).join(", ")));var M=i(6660);class B extends h{static featureName=M.t;constructor(r,n){let i=!(arguments.length>2&&void 0!==arguments[2])||arguments[2];super(r,n,M.t,i),this.skipNext=0;try{this.removeOnAbort=new AbortController}catch(e){}const o=this;o.ee.on("fn-start",(function(e,t,r){o.abortHandler&&(o.skipNext+=1)})),o.ee.on("fn-err",(function(t,r,n){o.abortHandler&&!n[M.A]&&((0,I.X)(n,M.A,(function(){return!0})),this.thrown=!0,(0,s.p)("err",[n,(0,p.z)()],void 0,e.D.jserrors,o.ee))})),o.ee.on("fn-end",(function(){o.abortHandler&&!this.thrown&&o.skipNext>0&&(o.skipNext-=1)})),o.ee.on("internal-error",(function(t){(0,s.p)("ierr",[t,(0,p.z)(),!0],void 0,e.D.jserrors,o.ee)})),this.origOnerror=c._A.onerror,c._A.onerror=this.onerrorHandler.bind(this),c._A.addEventListener("unhandledrejection",(t=>{const r=function(e){let t="Unhandled Promise Rejection: ";if(e instanceof Error)try{return e.message=t+e.message,e}catch(t){return e}if(void 0===e)return new Error(t);try{return new Error(t+(0,D.P)(e))}catch(e){return new Error(t)}}(t.reason);(0,s.p)("err",[r,(0,p.z)(),!1,{unhandledPromiseRejection:1}],void 0,e.D.jserrors,this.ee)}),(0,O.m$)(!1,this.removeOnAbort?.signal)),(0,k.gy)(this.ee),(0,k.BV)(this.ee),(0,k.em)(this.ee),(0,t.OP)(r).xhrWrappable&&(0,k.Kf)(this.ee),this.abortHandler=this.#e,this.importAggregator()}#e(){this.removeOnAbort?.abort(),this.abortHandler=void 0}onerrorHandler(t,r,n,i,o){"function"==typeof this.origOnerror&&this.origOnerror(...arguments);try{this.skipNext?this.skipNext-=1:(0,s.p)("err",[o||new F(t,r,n),(0,p.z)()],void 0,e.D.jserrors,this.ee)}catch(t){try{(0,s.p)("ierr",[t,(0,p.z)(),!0],void 0,e.D.jserrors,this.ee)}catch(e){}}return!1}}function F(e,t,r){this.message=e||"Uncaught error with no additional information",this.sourceURL=t,this.line=r}let U=1;const q="nr@id";function G(e){const t=typeof e;return!e||"object"!==t&&"function"!==t?-1:e===c._A?0:(0,I.X)(e,q,(function(){return U++}))}function V(e){if("string"==typeof e&&e.length)return e.length;if("object"==typeof e){if("undefined"!=typeof ArrayBuffer&&e instanceof ArrayBuffer&&e.byteLength)return e.byteLength;if("undefined"!=typeof Blob&&e instanceof Blob&&e.size)return e.size;if(!("undefined"!=typeof FormData&&e instanceof FormData))try{return(0,D.P)(e).length}catch(e){return}}}var X=i(7243);class W{constructor(e){this.agentIdentifier=e,this.generateTracePayload=this.generateTracePayload.bind(this),this.shouldGenerateTrace=this.shouldGenerateTrace.bind(this)}generateTracePayload(e){if(!this.shouldGenerateTrace(e))return null;var r=(0,t.DL)(this.agentIdentifier);if(!r)return null;var n=(r.accountID||"").toString()||null,i=(r.agentID||"").toString()||null,o=(r.trustKey||"").toString()||null;if(!n||!i)return null;var a=(0,_.M)(),s=(0,_.Ht)(),c=Date.now(),u={spanId:a,traceId:s,timestamp:c};return(e.sameOrigin||this.isAllowedOrigin(e)&&this.useTraceContextHeadersForCors())&&(u.traceContextParentHeader=this.generateTraceContextParentHeader(a,s),u.traceContextStateHeader=this.generateTraceContextStateHeader(a,c,n,i,o)),(e.sameOrigin&&!this.excludeNewrelicHeader()||!e.sameOrigin&&this.isAllowedOrigin(e)&&this.useNewrelicHeaderForCors())&&(u.newrelicHeader=this.generateTraceHeader(a,s,c,n,i,o)),u}generateTraceContextParentHeader(e,t){return"00-"+t+"-"+e+"-01"}generateTraceContextStateHeader(e,t,r,n,i){return i+"@nr=0-1-"+r+"-"+n+"-"+e+"----"+t}generateTraceHeader(e,t,r,n,i,o){if(!("function"==typeof c._A?.btoa))return null;var a={v:[0,1],d:{ty:"Browser",ac:n,ap:i,id:e,tr:t,ti:r}};return o&&n!==o&&(a.d.tk=o),btoa((0,D.P)(a))}shouldGenerateTrace(e){return this.isDtEnabled()&&this.isAllowedOrigin(e)}isAllowedOrigin(e){var r=!1,n={};if((0,t.Mt)(this.agentIdentifier,"distributed_tracing")&&(n=(0,t.P_)(this.agentIdentifier).distributed_tracing),e.sameOrigin)r=!0;else if(n.allowed_origins instanceof Array)for(var i=0;i 2&&void 0!==arguments[2])||arguments[2];super(r,n,Z.t,i),(0,t.OP)(r).xhrWrappable&&(this.dt=new W(r),this.handler=(e,t,r,n)=>(0,s.p)(e,t,r,n,this.ee),(0,k.u5)(this.ee),(0,k.Kf)(this.ee),function(r,n,i,o){function a(e){var t=this;t.totalCbs=0,t.called=0,t.cbTime=0,t.end=E,t.ended=!1,t.xhrGuids={},t.lastSize=null,t.loadCaptureCalled=!1,t.params=this.params||{},t.metrics=this.metrics||{},e.addEventListener("load",(function(r){_(t,e)}),(0,O.m$)(!1)),c.IF||e.addEventListener("progress",(function(e){t.lastSize=e.loaded}),(0,O.m$)(!1))}function s(e){this.params={method:e[0]},T(this,e[1]),this.metrics={}}function u(e,n){var i=(0,t.DL)(r);i.xpid&&this.sameOrigin&&n.setRequestHeader("X-NewRelic-ID",i.xpid);var a=o.generateTracePayload(this.parsedOrigin);if(a){var s=!1;a.newrelicHeader&&(n.setRequestHeader("newrelic",a.newrelicHeader),s=!0),a.traceContextParentHeader&&(n.setRequestHeader("traceparent",a.traceContextParentHeader),a.traceContextStateHeader&&n.setRequestHeader("tracestate",a.traceContextStateHeader),s=!0),s&&(this.dt=a)}}function d(e,t){var r=this.metrics,i=e[0],o=this;if(r&&i){var a=V(i);a&&(r.txSize=a)}this.startTime=(0,p.z)(),this.listener=function(e){try{"abort"!==e.type||o.loadCaptureCalled||(o.params.aborted=!0),("load"!==e.type||o.called===o.totalCbs&&(o.onloadCalled||"function"!=typeof t.onload)&&"function"==typeof o.end)&&o.end(t)}catch(e){try{n.emit("internal-error",[e])}catch(e){}}};for(var s=0;s 1?e[1]=i:e.push(i)}else e[0]&&e[0].headers&&s(e[0].headers,n)&&(this.dt=n);function s(e,t){var r=!1;return t.newrelicHeader&&(e.set("newrelic",t.newrelicHeader),r=!0),t.traceContextParentHeader&&(e.set("traceparent",t.traceContextParentHeader),t.traceContextStateHeader&&e.set("tracestate",t.traceContextStateHeader),r=!0),r}}function x(e,t){this.params={},this.metrics={},this.startTime=(0,p.z)(),this.dt=t,e.length>=1&&(this.target=e[0]),e.length>=2&&(this.opts=e[1]);var r,n=this.opts||{},i=this.target;"string"==typeof i?r=i:"object"==typeof i&&i instanceof Y?r=i.url:c._A?.URL&&"object"==typeof i&&i instanceof URL&&(r=i.href),T(this,r);var o=(""+(i&&i instanceof Y&&i.method||n.method||"GET")).toUpperCase();this.params.method=o,this.txSize=V(n.body)||0}function A(t,r){var n;this.endTime=(0,p.z)(),this.params||(this.params={}),this.params.status=r?r.status:0,"string"==typeof this.rxSize&&this.rxSize.length>0&&(n=+this.rxSize);var o={txSize:this.txSize,rxSize:n,duration:(0,p.z)()-this.startTime};i("xhr",[this.params,o,this.startTime,this.endTime,"fetch"],this,e.D.ajax)}function E(t){var r=this.params,n=this.metrics;if(!this.ended){this.ended=!0;for(var o=0;o 2&&void 0!==arguments[2])||arguments[2];super(e,t,we.t,r),this.importAggregator()}}new class{constructor(e){let t=arguments.length>1&&void 0!==arguments[1]?arguments[1]:(0,_.ky)(16);c._A?(this.agentIdentifier=t,this.sharedAggregator=new y({agentIdentifier:this.agentIdentifier}),this.features={},this.desiredFeatures=new Set(e.features||[]),this.desiredFeatures.add(m),Object.assign(this,(0,a.j)(this.agentIdentifier,e,e.loaderType||"agent")),this.start()):(0,l.Z)("Failed to initial the agent. Could not determine the runtime environment.")}get config(){return{info:(0,t.C5)(this.agentIdentifier),init:(0,t.P_)(this.agentIdentifier),loader_config:(0,t.DL)(this.agentIdentifier),runtime:(0,t.OP)(this.agentIdentifier)}}start(){const t="features";try{const r=n(this.agentIdentifier),i=[...this.desiredFeatures];i.sort(((t,r)=>e.p[t.featureName]-e.p[r.featureName])),i.forEach((t=>{if(r[t.featureName]||t.featureName===e.D.pageViewEvent){const n=function(t){switch(t){case e.D.ajax:return[e.D.jserrors];case e.D.sessionTrace:return[e.D.ajax,e.D.pageViewEvent];case e.D.sessionReplay:return[e.D.sessionTrace];case e.D.pageViewTiming:return[e.D.pageViewEvent];default:return[]}}(t.featureName);n.every((e=>r[e]))||(0,l.Z)("".concat(t.featureName," is enabled but one or more dependent features has been disabled (").concat((0,D.P)(n),"). This may cause unintended consequences or missing data...")),this.features[t.featureName]=new t(this.agentIdentifier,this.sharedAggregator)}})),(0,T.Qy)(this.agentIdentifier,this.features,t)}catch(e){(0,l.Z)("Failed to initialize all enabled instrument classes (agent aborted) -",e);for(const e in this.features)this.features[e].abortHandler?.();const r=(0,T.fP)();return delete r.initializedAgents[this.agentIdentifier]?.api,delete r.initializedAgents[this.agentIdentifier]?.[t],delete this.sharedAggregator,r.ee?.abort(),delete r.ee?.get(this.agentIdentifier),!1}}}({features:[J,m,S,class extends h{static featureName=oe;constructor(t,r){if(super(t,r,oe,!(arguments.length>2&&void 0!==arguments[2])||arguments[2]),!c.il)return;const n=this.ee;let i;(0,k.QU)(n),this.eventsEE=(0,k.em)(n),this.eventsEE.on(se,(function(e,t){this.bstStart=(0,p.z)()})),this.eventsEE.on(ae,(function(t,r){(0,s.p)("bst",[t[0],r,this.bstStart,(0,p.z)()],void 0,e.D.sessionTrace,n)})),n.on(ce+ne,(function(e){this.time=(0,p.z)(),this.startPath=location.pathname+location.hash})),n.on(ce+ie,(function(t){(0,s.p)("bstHist",[location.pathname+location.hash,this.startPath,this.time],void 0,e.D.sessionTrace,n)}));try{i=new PerformanceObserver((t=>{const r=t.getEntries();(0,s.p)(te,[r],void 0,e.D.sessionTrace,n)})),i.observe({type:re,buffered:!0})}catch(e){}this.importAggregator({resourceObserver:i})}},C,xe,B,class extends h{static featureName=de;constructor(e,r){if(super(e,r,de,!(arguments.length>2&&void 0!==arguments[2])||arguments[2]),!c.il)return;if(!(0,t.OP)(e).xhrWrappable)return;try{this.removeOnAbort=new AbortController}catch(e){}let n,i=0;const o=this.ee.get("tracer"),a=(0,k._L)(this.ee),s=(0,k.Lg)(this.ee),u=(0,k.BV)(this.ee),d=(0,k.Kf)(this.ee),f=this.ee.get("events"),l=(0,k.u5)(this.ee),h=(0,k.QU)(this.ee),g=(0,k.Gm)(this.ee);function m(e,t){h.emit("newURL",[""+window.location,t])}function v(){i++,n=window.location.hash,this[ve]=(0,p.z)()}function b(){i--,window.location.hash!==n&&m(0,!0);var e=(0,p.z)();this[pe]=~~this[pe]+e-this[ve],this[ye]=e}function y(e,t){e.on(t,(function(){this[t]=(0,p.z)()}))}this.ee.on(ve,v),s.on(be,v),a.on(be,v),this.ee.on(ye,b),s.on(ge,b),a.on(ge,b),this.ee.buffer([ve,ye,"xhr-resolved"],this.featureName),f.buffer([ve],this.featureName),u.buffer(["setTimeout"+le,"clearTimeout"+fe,ve],this.featureName),d.buffer([ve,"new-xhr","send-xhr"+fe],this.featureName),l.buffer([me+fe,me+"-done",me+he+fe,me+he+le],this.featureName),h.buffer(["newURL"],this.featureName),g.buffer([ve],this.featureName),s.buffer(["propagate",be,ge,"executor-err","resolve"+fe],this.featureName),o.buffer([ve,"no-"+ve],this.featureName),a.buffer(["new-jsonp","cb-start","jsonp-error","jsonp-end"],this.featureName),y(l,me+fe),y(l,me+"-done"),y(a,"new-jsonp"),y(a,"jsonp-end"),y(a,"cb-start"),h.on("pushState-end",m),h.on("replaceState-end",m),window.addEventListener("hashchange",m,(0,O.m$)(!0,this.removeOnAbort?.signal)),window.addEventListener("load",m,(0,O.m$)(!0,this.removeOnAbort?.signal)),window.addEventListener("popstate",(function(){m(0,i>1)}),(0,O.m$)(!0,this.removeOnAbort?.signal)),this.abortHandler=this.#e,this.importAggregator()}#e(){this.removeOnAbort?.abort(),this.abortHandler=void 0}}],loaderType:"spa"})})(),window.NRBA=o})(); window.jQuery || document.write(' ') CKEDITOR_BASEPATH='https://f1000research.com/js/vendor/ckeditor/' window.reactTheme = 'research'; window.MathJax = { CommonHTML: { linebreaks: { automatic: true } }, 'HTML-CSS': { linebreaks: { automatic: true } }, SVG: { linebreaks: { automatic: true } }, AuthorInit: function() { MathJax.Hub.Register.MessageHook('End Process', function () { let timeout = false; // holder for timeout id const delay = 250; // delay after event is "complete" to run callback const reflowMath = function() { const dispFormulas = document.querySelectorAll('.disp-formula.panel'); if (!dispFormulas) { return; } for (const dispFormula of dispFormulas) { const child = dispFormula.querySelector('.MathJax_Preview').nextSibling.firstChild; const isMultiline = MathJax.Hub.getAllJax(dispFormula)[0].root.isMultiline; if (dispFormula.offsetWidth < child.offsetWidth || isMultiline) { MathJax.Hub.Queue(['Rerender', MathJax.Hub, dispFormula]); } } }; window.addEventListener('resize', function() { clearTimeout(timeout); // clear the timeout timeout = setTimeout(reflowMath, delay); // start timing for event "completion" }); }); }, }; if (window.location.hash == '#_=_'){ window.location = window.location.href.split('#')[0] } !function(f,b,e,v,n,t,s){if(f.fbq)return;n=f.fbq=function() {n.callMethod? n.callMethod.apply(n,arguments):n.queue.push(arguments)} ;if(!f._fbq)f._fbq=n; n.push=n;n.loaded=!0;n.version='2.0';n.queue=[];t=b.createElement(e);t.async=!0; t.src=v;s=b.getElementsByTagName(e)[0];s.parentNode.insertBefore(t,s)}(window, document,'script','https://connect.facebook.net/en_US/fbevents.js'); fbq('init', '1641728616063202'); fbq('track', "PixelInitialized", {}); (function(h,o,t,j,a,r){ h.hj=h.hj||function(){(h.hj.q=h.hj.q||[]).push(arguments)}; h._hjSettings={hjid:2318163,hjsv:6}; a=o.getElementsByTagName('head')[0]; r=o.createElement('script');r.async=1; r.src=t+h._hjSettings.hjid+j+h._hjSettings.hjsv; a.appendChild(r); })(window,document,'https://static.hotjar.com/c/hotjar-','.js?sv='); search file_upload Submit your research search menu close search Browse Gateways & Collections How to Publish Submit your Research My Submissions Article Guidelines Article Guidelines (New Versions) Open Data, Software and Code Guidelines Open Data and Accessible Source Materials Guidelines (HSS) Open Data, Software and Code Guidelines (PSE) Prepublication Checks Production Process Posters and Slides Guidelines Document Guidelines Article Processing Charges Peer Review Finding Article Reviewers About How it Works For Reviewers Our Advisors Policies Glossary FAQs For Developers Newsroom Contact My Research Submissions Content and Tracking Alerts My Details Sign In file_upload Submit your research { "@context": "https://schema.org", "@type": "ScholarlyArticle", "mainEntityOfPage": { "@type": "WebPage", "@id": "https://f1000research.com/articles/6-577" }, "headline": "Validation of commercial ERK antibodies against the ERK orthologue of the scleractinian coral Stylophora...", "datePublished": "2017-04-26T15:38:00", "dateModified": "2017-07-03T13:51:41", "author": [ { "@type": "Person", "name": "Lucile Courtial" }, { "@type": "Person", "name": "Vincent Picco" }, { "@type": "Person", "name": "Gilles Pagès" }, { "@type": "Person", "name": "Christine Ferrier-Pagès" } ], "publisher": { "@type": "Organization", "name": "F1000Research", "logo": { "@type": "ImageObject", "url": "https://f1000research.com/img/AMP/F1000Research_image.png", "height": 480, "width": 60 } }, "image": { "@type": "ImageObject", "url": "https://f1000research.com/img/AMP/F1000Research_image.png", "height": 1200, "width": 150 }, "description": "The extracellular signal-regulated protein kinase (ERK) signalling pathway controls key cellular processes, such as cell cycle regulation, cell fate determination and the response to external stressors. Although ERK functions are well studied in a variety of living organisms ranging from yeast to mammals, its functions in corals are still poorly known. The present work aims to give practical tools to study the expression level of ERK protein and the activity of the ERK signalling pathway in corals. The antibody characterisation experiment was performed five times and identical results were obtained. The present study validated the immune-reactivity of commercially available antibodies directed against ERK and its phosphorylated/activated forms on protein extracts of the reef-building coral Stylophora pistillata." } { "@context": "http://schema.org", "@type": "BreadcrumbList", "itemListElement": [ { "@type": "ListItem", "position": "1", "item": { "@id": "https://f1000research.com/", "name": "Home" } }, { "@type": "ListItem", "position": "2", "item": { "@id": "https://f1000research.com/browse/articles", "name": "Browse" } }, { "@type": "ListItem", "position": "3", "item": { "@id": "https://f1000research.com/articles/6-577/v2", "name": "Validation of commercial ERK antibodies against the ERK orthologue..." } } ] } Home Browse Validation of commercial ERK antibodies against the ERK orthologue... ALL Metrics - Views Downloads Get PDF Get XML Cite How to cite this article Courtial L, Picco V, Pagès G and Ferrier-Pagès C. Validation of commercial ERK antibodies against the ERK orthologue of the scleractinian coral Stylophora pistillata [version 2; peer review: 3 approved] . F1000Research 2017, 6 :577 ( https://doi.org/10.12688/f1000research.11365.2 ) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. Close Copy Citation Details Export Export Citation Sciwheel EndNote Ref. Manager Bibtex ProCite Sente EXPORT Select a format first Track Share ▬ ✚ Antibody Validation Article Revised Validation of commercial ERK antibodies against the ERK orthologue of the scleractinian coral Stylophora pistillata [version 2; peer review: 3 approved] Lucile Courtial https://orcid.org/0000-0002-9396-1219 1-3 * , Vincent Picco https://orcid.org/0000-0001-5251-1521 4 * , Gilles Pagès https://orcid.org/0000-0002-8466-5610 4,5 , Christine Ferrier-Pagès 1 Lucile Courtial https://orcid.org/0000-0002-9396-1219 1-3 * , Vincent Picco https://orcid.org/0000-0001-5251-1521 4 * , Gilles Pagès https://orcid.org/0000-0002-8466-5610 4,5 , Christine Ferrier-Pagès 1 * Equal contributors PUBLISHED 03 Jul 2017 Author details Author details 1 Marine Department, Centre Scientifique de Monaco, Monaco, MC-98000, Monaco 2 Sorbonne Universités, Pierre and Marie Curie University, Paris, 75252, France 3 Laboratoire d’Excellence, UMR ENTROPIE, Nouméa, 98848, New Caledonia 4 Biomedical Department, Centre Scientifique de Monaco, Monaco, MC-98000, Monaco 5 Institute for Research on Cancer and Aging of Nice (IRCAN), University Nice Sophia-Antipolis, CNRS UMR7284/INSERM U1081, Centre Antoine Lacassagne, Nice, 06189, France Lucile Courtial Roles: Conceptualization, Data Curation, Formal Analysis, Investigation, Methodology, Writing – Original Draft Preparation, Writing – Review & Editing Vincent Picco Roles: Conceptualization, Data Curation, Formal Analysis, Investigation, Methodology, Project Administration, Supervision, Writing – Original Draft Preparation, Writing – Review & Editing Gilles Pagès Roles: Conceptualization, Resources, Writing – Original Draft Preparation Christine Ferrier-Pagès Roles: Conceptualization, Resources, Supervision, Writing – Original Draft Preparation OPEN PEER REVIEW DETAILS REVIEWER STATUS This article is included in the Antibody Validations gateway. Abstract The extracellular signal-regulated protein kinase (ERK) signalling pathway controls key cellular processes, such as cell cycle regulation, cell fate determination and the response to external stressors. Although ERK functions are well studied in a variety of living organisms ranging from yeast to mammals, its functions in corals are still poorly known. The present work aims to give practical tools to study the expression level of ERK protein and the activity of the ERK signalling pathway in corals. The antibody characterisation experiment was performed five times and identical results were obtained. The present study validated the immune-reactivity of commercially available antibodies directed against ERK and its phosphorylated/activated forms on protein extracts of the reef-building coral Stylophora pistillata . READ ALL READ LESS Keywords Antibody validation, ERK, Corals, MAPK Corresponding Author(s) Vincent Picco ( [email protected] ) Christine Ferrier-Pagès ( [email protected] ) Close Corresponding authors: Vincent Picco, Christine Ferrier-Pagès Competing interests: No competing interests were disclosed. Grant information: Financial support to CFP, GP, LC and VP was provided by the Centre Scientifique de Monaco and Paris VI University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Copyright: © 2017 Courtial L et al . This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication). How to cite: Courtial L, Picco V, Pagès G and Ferrier-Pagès C. Validation of commercial ERK antibodies against the ERK orthologue of the scleractinian coral Stylophora pistillata [version 2; peer review: 3 approved] . F1000Research 2017, 6 :577 ( https://doi.org/10.12688/f1000research.11365.2 ) First published: 26 Apr 2017, 6 :577 ( https://doi.org/10.12688/f1000research.11365.1 ) Latest published: 03 Jul 2017, 6 :577 ( https://doi.org/10.12688/f1000research.11365.2 ) Revised Amendments from Version 1 In order to comply with the referees' requests, we added an experiment consisting of inducing ERK activity by exposing corals to UV radiation, thermal stress or a combination of both. We added new figures as well as their corresponding text in the Methods and the Results sections. Specifically, in order to answer one of the referees' concerns, we have slightly modified Figure 2 and Supplementary Figure S1 by adding the amido black total protein stainings of the western blot membranes as a loading control. We have also added a new Figure 3 and the corresponding Supplementary Figure S2 (full length images of the western blot). In order to comply with the referees' requests, we added an experiment consisting of inducing ERK activity by exposing corals to UV radiation, thermal stress or a combination of both. We added new figures as well as their corresponding text in the Methods and the Results sections. Specifically, in order to answer one of the referees' concerns, we have slightly modified Figure 2 and Supplementary Figure S1 by adding the amido black total protein stainings of the western blot membranes as a loading control. We have also added a new Figure 3 and the corresponding Supplementary Figure S2 (full length images of the western blot). See the authors' detailed response to the review by Andrea Pitzschke See the authors' detailed response to the review by Immacolata Castellano See the authors' detailed response to the review by María L. Parages READ REVIEWER RESPONSES Introduction Mitogen activated protein kinases (MAPKs) are highly conserved proteins involved in signalling pathways and control key cellular processes such as proliferation, differentiation, migration, survival and apoptosis ( Dhillon et al ., 2007 ). The MAPK gene family encompasses three major subfamilies: the extracellular signal-regulated kinase (ERK), p38/HOG and c-Jun N-terminal kinase (JNK) groups. The ERK family is the most studied in mammals ( Boulton et al ., 1990 ; Dhillon et al ., 2007 ) because it is involved in meiosis, mitosis and post mitotic functions in differentiated cells, as well as in the oxidative stress response and wound healing ( Castellano et al. , 2014 ; Johnson & Lapadat, 2002 ; Matsubayashi et al ., 2004 ; Runchel et al ., 2011 ). The ERK gene family is evolutionnarily conserved and is found in all eukaryotes, including yeasts, plants, vertebrates and invertebrates ( Chen et al ., 2001 ; Widmann et al ., 1999 ). Although recent molecular studies have shown the existence of ERK genes in different coral species ( Mayfield et al ., 2010 ; Siboni et al ., 2012 ; van de Water et al ., 2015 ), ERK activity and specific functions are not yet clearly defined. ERK activation occurs through phosphorylation of the Threonine and Tyrosine residues of an ERK-specific TEY motif by the upstream kinases of ERK, the mitogen-activated protein kinase kinase (MAPKK or MEK). ERK phosphorylation on these residues is classically considered the most appropriate readout for the activity of the ERK signalling pathway. However, it has never been monitored in corals. Overall, MAPK activities in corals have only been investigated once, in a study focusing on the JNK subfamily ( Courtial et al ., 2017 ). In this work, we used the scleractinian coral Stylophora pistillata , a very abundant species in most tropical reefs ( Veron & Stafford-Smith, 2000 ). We applied the same protocol as in Courtial et al . (2017) to demonstrate the efficiency of antibodies directed against the mammalian phosphorylated forms of ERK (pERK) and total ERK to detect the ERK orthologs in S. pistillata ( Table 1 ). According to the manufacturer’s instructions, the antibody used in this study and directed against the Thr202/Tyr204 di-phosphorylated active ERK (Thermo Scientific Pierce; MA5-15174) showed reactivity with fruit fly, human, mink, mouse, non-human primate, pig, rat and zebrafish. The immunogen used to generate this rabbit IgG monoclonal antibody was a synthetic phosphopeptide corresponding to residues surrounding the phospho-Thr202/Tyr204 of the human p44/ERK1 MAP kinase. This antibody is not cross-reactive with the corresponding phosphorylated residues of either JNK/SAPK or p38. The ERK1/ERK2 antibody (Thermo Scientific Pierce; MA5-15605) used in the study previously showed reactivity with human and mouse samples. The immunogen used to generate this mouse IgG2b monoclonal antibody was a purified recombinant fragment of human MAPK. Table 1. Primary and secondary antibodies. Antibody Manufacturer Catalogue number RRID Concentration p44+42 MAPK (Erk1,2) Antibody (3F8) Thermo Fisher Scientific MA5-15605 AB_10983247 1µg/mL (1/1000) Phospho-p44 MAPK + p42 MAPK pTyr204 Antibody (B.742.5) Thermo Fisher Scientific MA5-15174 AB_10980347 1µg/mL (1/1000) Peroxidase-AffiniPure Goat Anti-Mouse IgG (H+L) antibody Jackson ImmunoResearch Labs 115-035-003 AB_10015289 1µg/mL (1/10000) Peroxidase-AffiniPure Goat Anti-Rabbit IgG (H+L) antibody Jackson ImmunoResearch Labs 111-035-003 AB_2313567 1µg/mL (1/10000) IRDye 800CW Goat Anti-Rabbit IgG (H+L) LI-COR Biosciences 926-32211 AB_621843 0.1µg/mL (1/10000) IRDye 680RD Goat anti-Mouse IgG (H + L) LI-COR Biosciences 926-68070 AB_10956588 0.1µg/mL (1/10000) Methods Maintenance of Stylophora pistillata nubbins and human fibroblasts Nubbins of Stylophora pistillata were collected from five mother colonies maintained in the aquaria facilities of the Centre Scientifique de Monaco. Two small nubbins (3–5 cm long) were cut off from each mother colony, and were allowed to heal for four weeks in 15 L open system tanks before the experiments. Corals were maintained in the same conditions as the mother colonies, i.e. at 25°C, under a photosynthetic active radiation of 200 µmol photon.m -2 .s -1 provided by 400 W metal halide lamps (HPIT, Philips) and were fed twice a week with Artemia salina nauplii . Seawater in the tanks was continuously renewed at a rate of 10 L.h -1 . Immortalized skin fibroblasts (BJ-EHLT cells) were kindly provided by E. Gilson’s lab (IRCAN) and cultured in Dulbecco’s Modified Eagle’s Medium (Invitrogen, Villebon-sur-Yvette, France) supplemented with 10% heat-inactivated fetal calf serum (Dutscher, Brumath, France) at 37°C in an atmosphere of 5% CO 2 , as previously described ( Biroccio et al ., 2013 ). UO126 treatment of coral nubbins Incubations were performed in 100 mL beakers containing one coral nubbin each, and filled with 40 mL of 0.45 μm filtered seawater. They were placed in the dark for one hour in either a control condition containing 0.005% DMSO (vehicle) or a condition with 5 μmol.L -1 UO126 (Selleck Chemicals), a MEK inhibitor ( Tang et al ., 2003 ). The incubation medium was continuously stirred using magnetic stirrers at a constant temperature of 25°C. At the end of the incubation, nubbins were frozen and kept at – 80°C prior to western blot analysis. UVR and temperature treatment of coral nubbins Incubations were performed in 100 mL beakers containing one coral nubbin each and filled with 40 mL of 0.45 μm filtered seawater and continuously stirred using magnetic stirrers. High temperature or/and ultraviolet radiation (UVR) stresses (i.e. four environmental conditions: control (at 25°C and without UVR), thermal stress (30°C without UVR), UVR stress (25°C under UVR), thermal and UVR stresses (30°C and under UVR)) were applied to corals and ERK activation was monitored after 30 minutes of stress. Thermal stress corresponded to an increase in temperature from the normal culture condition of 25°C to 30°C. The UVR stress corresponded to an increase in UVR from 0 (HQI lamps in the culture conditions) to a radiation intensity of about 3 W.m −2 UVB and 30 W.m −2 UVA (Q-Panel UVA 340 lamps). At the end of the incubation, nubbins were frozen and kept at – 80°C prior to western blot analysis. Western blot analysis Immuno-detections were performed as in Courtial et al. (2017 ; Table 2 and Table 3 ). Briefly, nubbins were airbrushed in 1 mL Laemmli buffer (2% SDS, 10% glycerol, 50mM Tris HCL pH7), ( Laemmli, 1970 ) using an air-pick (5 bars) to remove the totality of the tissues surrounding the skeleton was removed from coral. Samples were then sonicated for 30 seconds, and centrifuged (3 × 5 minutes at 15 000 g) to remove the lipid supernatant and debris. Fibroblasts were washed twice in phosphate buffered saline solution (PBS), lyzed in Laemmli buffer directly in the dishes and sonicated for 30 seconds. The total protein concentration of all samples was determined using a BCA protein Assay Kit (Thermo Fisher Scientific), according to the manufacturer’s recommendation. 1,4 Dithiothreitol (1 mM) and bromophenol blue (0.1%) were added to the samples, which were then heated for 5 minutes at 95°C. Table 2. Tissue extraction and western blot protocol. Process Reagent Manufacturer Catalogue number Concentration/Composition Tissue extraction Laemmli buffer 1.5X Homemade 150 mM Tris-HCl pH 7, 25% glycerol, 2% SDS Sample preparation Laemmli - 1,4 Dithiothreitol - bromophenol blue solution Homemade 1.5 X- 50 mM - 0.1% Electrophoresis ECL gradient gel 8–16% GE Healthcare Lifesciences 29-9901-58 TG-SDS 10X running buffer EUROMEDEX EU0510 1X Protein transfer DUNN transfer buffer Homemade 10 mM NaHCO3 - 3 mM Na2CO3 - 10% Ethanol (pH 9.9) Blocking Blocking reagent Homemade PBS + milk (3%) Washes Wash buffer 10X Homemade PBS 10X Tween 20 1N Membrane coloration Coloration buffer Homemade Isopropanol (25%) + acetic acid (10%) + amido black (0.1%) Membrane destain Destain buffer Homemade Isopropanol (25%) + acetic acid (10%) Target detection Immobilon Western HRP Substrate Millipore WBKLS0500 Reagents BCA QuantiPro BCA Assay Kit Sigma-Aldrich QPBCA-1KT Milk Itambe ® Methanol Sigma-Aldrich Table 3. Reagents for tissue extraction and western blots. Protocol steps Reagent Time Temperature Tissue extraction Laemmli 1.5 X (1mL) RT Sonication Laemmli 1.5 X 30 sec RT Centrifugation (x3 15000 g) Laemmli 1.5 X 3 × 5 min RT Addition of 1,4 Dithiothreitol - bromophenol blue solution Laemmli 1.5 X RT Heat up 5 min 95°C Electrophoresis (100 V) Running buffer variable RT Transfer (200 mA) Transfer buffer overnight 4°C Coloration Isopropanol (25%) + acetic acid (10%) + amido black (0.1%) 5 min RT Destain Isopropanol (25%) + acetic acid (10%) 3 × 5 min RT Blocking PBS + milk (3%) 30 min RT Primary antibodies PBS + milk (1%) + ab (1/1000) overnight 4°C Washes (3 times) Wash buffer 1X 3 × 15 min RT Secondary antibody PBS + milk (1%) + ab (1/10000) 2h RT Washes (5 times) Wash buffer 1X 5 × 15 min RT Detection Immobilon Western HRP Substrate 30 sec – 10 min RT 60 μg of proteins were separated on 10% polyacrylamide gels at 300 mA and 110 V at room temperature. Proteins were then transferred on a PVDF membrane at 4°C overnight in Dunn’s transfer buffer at 200 mA. After a rinse in distilled water, membranes were saturated for 30 minutes in PBS - 3% low fat milk, rinsed in PBS and incubated with primary antibodies diluted in PBS - 1% low fat milk at 4°C overnight. The antibody directed against Thr202/Tyr204 di-phosphorylated active ERK was from Thermo Scientific Pierce (rabbit monoclonal; MA5-15174; batch no. OC1680806); the anti-ERK1/2 antibody was from Thermo Scientific Pierce (mouse monoclonal; MA5-15605; batch no. PH1895491). After extensive washing (4×30 minutes) in PBS – 0.1% Tween 20, membranes were incubated for 2 hours at room temperature in the simultaneous presence of IRDye 680RD goat anti-mouse (925-68070) and IRDye 800CW goat anti-rabbit (925-32211; Li-COR Biotechnology GmbH, Bad Homburg, Germany) secondary antibodies, or with anti-mouse and anti-rabbit HRP-conjugated antibody. Another set of extensive rinsing (4×30 minutes) in PBS – 0.1% Tween 20 was performed before membranes were imaged with an Odyssey device (LI-COR Biosciences, Lincoln, Nebraska) to detect fluorescence and HRP activity using Millipore ECL. Densitometric analysis of the western blots was performed using Image Studio v2.1 software (Li-COR Biosciences). Intensity of the pERK signal was normalized to the intensity of ERK signal. The relative intensities between control and inhibitor conditions were compared using a t-test. Statistical analysis was done using the software Graphpad Prism v5.03. Results and discussion In order to confirm the presence of an ERK ortholog in corals, the human protein sequence of ERK1 (NP_001035145) was compared to the transcriptome database of Stylophora pistillata using the BLAST software ( Altschul et al. , 1990 ; Karako-Lampert et al ., 2014 ). An open reading frame was retrieved from the best hit sequence with a predicted molecular weight of 42 kDa ( Spi_isotig05348 ). This sequence (hereafter referred to as Spi-ERK for S. pistillata ERK) is the only one that shows an homology as high as 81%, 80% and 78% with the protein sequences of the cnidarians Nematostella vectensis ERK (Nv-ERK; XP_001629498.1), Hydra vulgaris ERK (Hv-ERK; XP_002154499.3) and the human MAPK3/ERK1 (Hs-ERK1), respectively ( Figure 1 ) ( Krishna et al ., 2013 ; Putnam et al ., 2007 ). These sequences all contain both the conserved kinase domains ( Hanks & Hunter, 1995 ) and the TEY motif of the catalytic domain, which is unique for ERK orthologs ( Davis, 2000 ; Figure 1 ). An interesting point to note is that a unique sequence showing these features is present in N. vectensis and H. vulgaris genomes, as well as in the S. pistillata transcriptome database. This result suggests that a single ortholog of ERK is present in these cnidarians, consistently with previous work where only one ERK ortholog was found ( Castellano et al. , 2014 ; Russo et al. , 2004 ) but as opposed to the two genes encoding ERKs in most mammalian genomes ( Ip & Davis, 1998 ). Furthermore, based on the high level of sequence conservation between distant species ( Hanks & Hunter, 1995 ), antibodies directed against portions of the ERK human proteins may recognize ERKs from other species. Accordingly, we detected a single immune-reactive band with the total-ERK antibody by western blot on S. pistillata extracts ( Figure 2A and Supplementary Figure S1 ). Spi-ERK should retain the mechanism of activation by phosphorylation of the Threonine and the Tyrosine residues of the ERK-specific TEY motif. Hence, the MA5-15174 antibody directed against the phosphorylated Thr202 and the Tyr204 ( i.e. the phosphorylated TEY motif) should detect a phosphorylated TEY motif of Spi-ERK (phospho-ERK). This is consistent with what we observed, as we detected a unique immune-reactive band of approximately 40 kDa with both antibodies ( Figure 2A ). Figure 1. Sequence alignment of MAPK orthologs. The ERK orthologs of Stylphora pistillata (Spi-ERK), Nematostella vectensis (Nv-ERK), Hydra vulgaris (Hv-ERK), and the human ERK1 (Hs-ERK1) protein sequences are shown. The ERK-specific TEY motif is highlighted in red. The eleven conserved kinase domains are underlined. Figure 2. Detection of ERK activity in corals. ( A ) Fluorescent immunoblot revealing activated (pERK) and total forms of ERK (ERK) present in Stylphora pistillata nubbins. Molecular weight standards in kilo Daltons (kDa) are indicated on the left side of the figure. ( B ) Immunoblot performed with ERK and pERK antibodies on protein extracts from coral nubbins incubated in the absence (Control) or presence of the MEK inhibitor U0126. Densitometric analysis of activated ERK intensities is presented on the right of the figure. The amido black total protein staining of the western blot membrane is shown as a loading control. The medians and standard deviations of three independent experiments are presented (***, p<0.01, t-test). Interestingly, the fluorescent immunoblot technique showed that the bands detected with the phosphorylated- and the total-ERK antibodies mostly co-migrate, suggesting that the same protein is detected ( Figure 2A ). The slight electrophoretic migration shift of the band detected with the anti-phosphorylated ERK antibody would be consistent with the phosphorylation of the threonine and tyrosine residues of the TEY motif as previously described ( Aoki et al ., 2011 ). These results suggest that ERK and its phosphorylated form are correctly recognized by the antibodies. RNAi interference techniques are not available in coral, and the confirmation that the immune reactive bands observed here specifically correspond to ERK could not be obtained through this method. In order to test the specificity of the antibodies, we therefore used U0126, a very potent and selective inhibitor of MEK ( Bain et al ., 2007 ). The limited thickness of the animal tissue covering the skeleton and the very large surface of contact of both ectoderm and endoderm with the seawater render S. pistillata suitable for treatment with drugs directly diluted in the seawater as we previously showed ( Courtial et al. , 2017 ). U0126 was previously shown to efficiently block MEK activity in a wide variety of organisms, including cnidarians ( Hasse et al ., 2014 ; Picco et al ., 2007 ; Röttinger et al. , 2004 ). When the inhibitor was added to the seawater, the intensity of the band detected by the anti-total ERK did not vary, while the intensity of the band detected with the anti-phosphorylated ERK antibody was significantly reduced ( Figure 2B and Supplementary Figure S1 ). Altogether, our results strongly suggest that the proteins detected with the two antibodies were ERK and pERK. To confirm that Spi-ERK activity can dynamically respond to changes in experimental conditions, we performed an induction experiment by modifying culture conditions of the corals. Courtial et al. (2017) showed that thermal and UVR stresses induced the formation of reactive oxygen species which are known to trigger ERK phosphorylation ( McCubrey et al. , 2006 ). ERK phosphorylation was enhanced in corals exposed to UVR, high temperature or a combination of both ( Figure 3 and Supplementary Figure S2 ). These results confirm that the antibodies characterized herein can be used to monitor ERK activity in corals. Finally, to assess the performance of these antibodies, we compared the signal obtained on S. pistillata and human fibroblasts protein extracts ( Figure 4 and Supplementary Figure S3 ). We loaded on the same gel 10µg of fibroblast total protein extract and different amounts of S. pistillata extracts (ranging from 80 to 10 µg). A signal comparable to the one obtained with the fibroblast extract was observed using 40 µg of coral proteins for both antibodies. This suggests that the affinity of the antibodies towards the coral proteins may be lower than for their human counterparts. Figure 3. Induction of Spi-ERK phosphorylation by thermal and UV stresses. Immunoblot performed with ERK and pERK antibodies on protein extracts from coral nubbins incubated for 30 minutes in control (Cont.), thermal stress (T), UV stress (UV) or a combination of thermal and UV stresses (UV + T) conditions. The amido black total protein staining of the western blot membrane is shown as a loading control. Figure 4. Relative sensitivities of ERK antibodies toward the human and coral proteins. Immunoblot performed with anti-ERK and anti-phospho-ERK on total protein extracts of human fibroblasts (BJ) and Stylphora pistillata . The amount of protein loaded in each lane is indicated on the Supplementary Figure S4. Dataset 1. Supplementary Figure S1. Uncropped blot images for Figure 2 and supplementary replicates. http://dx.doi.org/10.5256/f1000research.11365.d159188 ( A ) Biological replicates of fluorescent immunoblots performed in control conditions (Ct) are shown (Replicates 1 and 2). The portions of the images used in the main text are outlined. ( B ) Biological replicates of immunoblots performed on protein extracts from coral nubbins incubated in the absence (Control) or presence of the MEK inhibitor U0126 (UO) (Replicates 1 to 5). The amido black total protein staining of the western blot membrane is shown as a loading control. The portions of the images used in the main text are outlined. Dataset 2. Supplementary Figure S2. Uncropped blot images for Figure 3 and supplementary replicates. http://dx.doi.org/10.5256/f1000research.11365.d166821 The portions of the images used in the main text are outlined. Dataset 3. Supplementary Figure S3. Uncropped blot images for Figure 4 and supplementary replicates. http://dx.doi.org/10.5256/f1000research.11365.d166825 The portions of the images used in the main text are outlined. Conclusion This work showed that MA5-15174 and MA5-15605 are two specific antibodies that can be used to quantitatively assess Stylophora pistillata ERK phosphorylation/activity in different experimental or environmental conditions. We demonstrated the specificity of these antibodies and their good affinity towards their coral targets. It therefore provides the coral research community with a potent tool for the analysis of the activity of a signalling pathway involved in a wide variety of biological processes. Data availability Supplementary Figure S1. Uncropped blot images for Figure 2 and supplementary replicates. ( A ) Biological replicates of fluorescent immunoblots performed in control conditions (Ct) are shown (Replicates 1 and 2). The portions of the images used in the main text are outlined. ( B ) Biological replicates of immunoblots performed on protein extracts from coral nubbins incubated in the absence (Control) or presence of the MEK inhibitor U0126 (UO) (Replicates 1 to 5). The portions of the images used in the main text are outlined. doi, 10.5256/f1000research.11365.d159188 ( Courtial et al. , 2017a ). Supplementary Figure S2. Uncropped blot images for Figure 3 and supplementary replicates. The portions of the images used in the main text are outlined. doi, 10.5256/f1000research.11365.d166821 ( Courtial et al. , 2017b ). Supplementary Figure S3. Uncropped blot images for Figure 4 and supplementary replicates. The portions of the images used in the main text are outlined. doi, 10.5256/f1000research.11365.d166825 ( Courtial et al. , 2017c ). Author contributions CFP, GP, LC and VP conceived and designed the experiments. LC and VP performed the experiments. CFP, GP, LC and VP analyzed the data. CFP and GP contributed reagents/materials/analysis tools. CFP, GP, LC and VP wrote the paper. Competing interests No competing interests were disclosed. Grant information Financial support to CFP, GP, LC and VP was provided by the Centre Scientifique de Monaco and Pierre and Marie Curie University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Acknowledgements The authors thank Y. Cormerais for his help in the experimental setup, N. Caminiti-Seconds for the first coral extracts and assays as well as Pr. Denis Allemand, Director of the Centre Scientifique de Monaco for scientific support. Faculty Opinions recommended References Altschul SF, Gish W, Miller W, et al. : Basic local alignment search tool. J Mol Biol. 1990; 215 (3): 403–410. PubMed Abstract | Publisher Full Text Aoki K, Yamada M, Kunida K, et al. : Processive phosphorylation of ERK MAP kinase in mammalian cells. Proc Natl Acad Sci U S A. 2011; 108 (31): 12675–12680. PubMed Abstract | Publisher Full Text | Free Full Text Bain J, Plater L, Elliott M, et al. : The selectivity of protein kinase inhibitors: a further update. Biochem J. 2007; 408 (3): 297–315. PubMed Abstract | Publisher Full Text | Free Full Text Biroccio A, Cherfils-Vicini J, Augereau A, et al. : TRF2 inhibits a cell-extrinsic pathway through which natural killer cells eliminate cancer cells. Nat Cell Biol. 2013; 15 (7): 818–828. PubMed Abstract | Publisher Full Text Boulton TG, Yancopoulos GD, Gregory JS, et al. : An insulin-stimulated protein kinase similar to yeast kinases involved in cell cycle control. Science. 1990; 249 (4964): 64–67. PubMed Abstract | Publisher Full Text Castellano I, Ercolesi E, Palumbo A: Nitric oxide affects ERK signaling through down-regulation of MAP kinase phosphatase levels during larval development of the ascidian Ciona intestinalis . PLoS One. 2014; 9 (7): e102907. PubMed Abstract | Publisher Full Text | Free Full Text Chen Z, Gibson TB, Robinson F, et al. : MAP kinases. Chem Rev. 2001; 101 (8): 2449–2476. PubMed Abstract | Publisher Full Text Courtial L, Picco V, Gorver R, et al. : The c-Jun N-terminal kinase prevents oxidative stress induced by UV and thermal stresses in corals and human cells. Sci Rep. 2017; 7 : 45713. PubMed Abstract | Publisher Full Text | Free Full Text Courtial L, Picco V, Pagès G, et al. : Dataset 1 in: Validation of commercial ERK antibodies against the ERK orthologue of the scleractinian coral Stylophora pistillata . F1000Research. 2017a. Data Source Courtial L, Picco V, Pagès G, et al. : Dataset 2 in: Validation of commercial ERK antibodies against the ERK orthologue of the scleractinian coral Stylophora pistillata . F1000Research. 2017b. Data Source Courtial L, Picco V, Pagès G, et al. : Dataset 3 in: Validation of commercial ERK antibodies against the ERK orthologue of the scleractinian coral Stylophora pistillata . F1000Research. 2017c. Data Source Davis RJ: Signal transduction by the JNK group of MAP kinases. Cell. 2000; 103 (2): 239–252. PubMed Abstract | Publisher Full Text Dhillon AS, Hagan S, Rath O, et al. : MAP kinase signalling pathways in cancer. Oncogene. 2007; 26 (22): 3279–90. PubMed Abstract | Publisher Full Text Hanks SK, Hunter T: Protein kinases 6. The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification. FASEB J. 1995; 9 (8): 576–596. PubMed Abstract Hasse C, Holz O, Lange E, et al. : FGFR-ERK signaling is an essential component of tissue separation. Dev Biol. 2014; 395 (1): 154–166. PubMed Abstract | Publisher Full Text Ip YT, Davis RJ: Signal transduction by the c-Jun N-terminal kinase (JNK)--from inflammation to development. Curr Opin Cell Biol. 1998; 10 (2): 205–219. PubMed Abstract | Publisher Full Text Johnson GL, Lapadat R: Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases. Science. 2002; 298 (5600): 1911–1912. PubMed Abstract | Publisher Full Text Karako-Lampert S, Zoccola D, Salmon-Divon M, et al. : Transcriptome analysis of the scleractinian coral Stylophora pistillata . PLoS One. 2014; 9 (2): e88615. PubMed Abstract | Publisher Full Text | Free Full Text Krishna S, Nair A, Cheedipudi S, et al. : Deep sequencing reveals unique small RNA repertoire that is regulated during head regeneration in Hydra magnipapillata . Nucleic Acids Res. 2013; 41 (1): 599–616. PubMed Abstract | Publisher Full Text | Free Full Text Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970; 227 (5259): 680–685. PubMed Abstract | Publisher Full Text Matsubayashi Y, Ebisuya M, Honjoh S, et al. : ERK activation propagates in epithelial cell sheets and regulates their migration during wound healing. Curr Biol. 2004; 14 (8): 731–735. PubMed Abstract | Publisher Full Text Mayfield AB, Hsiao YY, Fan TY, et al. : Evaluating the temporal stability of stress-activated protein kinase and cytoskeleton gene expression in the Pacific reef corals Pocillopora damicornis and Seriatopora hystrix . J Exp Mar Bio Ecol. 2010; 395 (1–2): 215–222. Publisher Full Text McCubrey JA, LaHair MM, Franklin RA: Reactive oxygen species-induced activation of the MAP kinase signaling pathways. Antioxid Redox Signal. 2006; 8 (9–10): 1775–1789. PubMed Abstract | Publisher Full Text Picco V, Hudson C, Yasuo H: Ephrin-Eph signalling drives the asymmetric division of notochord/neural precursors in Ciona embryos. Development. 2007; 134 (8): 1491–1497. PubMed Abstract | Publisher Full Text Putnam NH, Srivastava M, Hellsten U, et al. : Sea Anemone Genome Reveals Ancestral Eumetazoan Gene Repertoire and Genomic Organization. Science. 2007; 317 (5834): 86–94. PubMed Abstract | Publisher Full Text Röttinger E, Besnardeau L, Lepage T: A Raf/MEK/ERK signaling pathway is required for development of the sea urchin embryo micromere lineage through phosphorylation of the transcription factor Ets. Development. 2004; 131 (5): 1075–1087. PubMed Abstract | Publisher Full Text Runchel C, Matsuzawa A, Ichijo H: Mitogen-activated protein kinases in mammalian oxidative stress responses. Antioxid Redox Signal. 2011; 15 (1): 205–218. PubMed Abstract | Publisher Full Text Russo GL, Tosto M, Mupo A, et al. : Biochemical and functional characterization of protein kinase CK2 in Ascidian Ciona intestinalis oocytes at fertilization. Cloning and sequence analysis of cDNA for alpha and beta subunits. J Biol Chem. 2004; 279 (31): 33012–33023. PubMed Abstract | Publisher Full Text Siboni N, Abrego D, Seneca F, et al. : Using bacterial extract along with differential gene expression in Acropora millepora Larvae to decouple the processes of attachment and metamorphosis. PLoS One. 2012; 7 (5): e37774. PubMed Abstract | Publisher Full Text | Free Full Text Tang QQ, Otto TC, Lane MD: Mitotic clonal expansion: A synchronous process required for adipogenesis. Proc Natl Acad Sci U S A. 2003; 100 (1): 44–49. PubMed Abstract | Publisher Full Text | Free Full Text van de Water JA, Leggat W, Bourne DG, et al. : Elevated seawater temperatures have a limited impact on the coral immune response following physical damage. Hydrobiologia. 2015; 759 (1): 201–214. Publisher Full Text Veron JE, Stafford-Smith M: Corals of the World. Volumes 1–3. Aust Inst Mar Sci. Townsville, Aust, 2000. Reference Source Widmann C, Gibson S, Jarpe MB, et al. : Mitogen-activated protein kinase: conservation of a three-kinase module from yeast to human. Physiol Rev. 1999; 79 (1): 143–180. PubMed Abstract Comments on this article Comments (0) Version 2 VERSION 2 PUBLISHED 26 Apr 2017 ADD YOUR COMMENT Comment Author details Author details 1 Marine Department, Centre Scientifique de Monaco, Monaco, MC-98000, Monaco 2 Sorbonne Universités, Pierre and Marie Curie University, Paris, 75252, France 3 Laboratoire d’Excellence, UMR ENTROPIE, Nouméa, 98848, New Caledonia 4 Biomedical Department, Centre Scientifique de Monaco, Monaco, MC-98000, Monaco 5 Institute for Research on Cancer and Aging of Nice (IRCAN), University Nice Sophia-Antipolis, CNRS UMR7284/INSERM U1081, Centre Antoine Lacassagne, Nice, 06189, France Lucile Courtial Roles: Conceptualization, Data Curation, Formal Analysis, Investigation, Methodology, Writing – Original Draft Preparation, Writing – Review & Editing Vincent Picco Roles: Conceptualization, Data Curation, Formal Analysis, Investigation, Methodology, Project Administration, Supervision, Writing – Original Draft Preparation, Writing – Review & Editing Gilles Pagès Roles: Conceptualization, Resources, Writing – Original Draft Preparation Christine Ferrier-Pagès Roles: Conceptualization, Resources, Supervision, Writing – Original Draft Preparation Competing interests No competing interests were disclosed. Grant information Financial support to CFP, GP, LC and VP was provided by the Centre Scientifique de Monaco and Paris VI University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Article Versions (2) version 2 Revised Published: 03 Jul 2017, 6:577 https://doi.org/10.12688/f1000research.11365.2 version 1 Published: 26 Apr 2017, 6:577 https://doi.org/10.12688/f1000research.11365.1 Copyright © 2017 Courtial L et al . This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication). Download Export To Sciwheel Bibtex EndNote ProCite Ref. Manager (RIS) Sente metrics Views Downloads F1000Research - - PubMed Central info_outline Data from PMC are received and updated monthly. - - Citations open_in_new 0 open_in_new 0 open_in_new SEE MORE DETAILS CITE how to cite this article Courtial L, Picco V, Pagès G and Ferrier-Pagès C. Validation of commercial ERK antibodies against the ERK orthologue of the scleractinian coral Stylophora pistillata [version 2; peer review: 3 approved] . F1000Research 2017, 6 :577 ( https://doi.org/10.12688/f1000research.11365.2 ) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS track receive updates on this article Track an article to receive email alerts on any updates to this article. TRACK THIS ARTICLE Share Open Peer Review Current Reviewer Status: ? Key to Reviewer Statuses VIEW HIDE Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Version 2 VERSION 2 PUBLISHED 03 Jul 2017 Revised Views 0 Cite How to cite this report: Parages ML. Reviewer Report For: Validation of commercial ERK antibodies against the ERK orthologue of the scleractinian coral Stylophora pistillata [version 2; peer review: 3 approved] . F1000Research 2017, 6 :577 ( https://doi.org/10.5256/f1000research.13040.r23990 ) The direct URL for this report is: https://f1000research.com/articles/6-577/v2#referee-response-23990 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 21 Jul 2017 María L. Parages , Department of Ecology, Faculty of Sciences, University of Málaga, Málaga, Spain Approved VIEWS 0 https://doi.org/10.5256/f1000research.13040.r23990 After author response, I recommend the manuscript for ... Continue reading READ ALL After author response, I recommend the manuscript for indexing. So I suggest the new status as Approved. Competing Interests: No competing interests were disclosed. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Parages ML. Reviewer Report For: Validation of commercial ERK antibodies against the ERK orthologue of the scleractinian coral Stylophora pistillata [version 2; peer review: 3 approved] . F1000Research 2017, 6 :577 ( https://doi.org/10.5256/f1000research.13040.r23990 ) The direct URL for this report is: https://f1000research.com/articles/6-577/v2#referee-response-23990 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Respond or Comment COMMENT ON THIS REPORT Views 0 Cite How to cite this report: Pitzschke A. Reviewer Report For: Validation of commercial ERK antibodies against the ERK orthologue of the scleractinian coral Stylophora pistillata [version 2; peer review: 3 approved] . F1000Research 2017, 6 :577 ( https://doi.org/10.5256/f1000research.13040.r24020 ) The direct URL for this report is: https://f1000research.com/articles/6-577/v2#referee-response-24020 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 04 Jul 2017 Andrea Pitzschke , Department of Cell Biology and Physiology, University of Salzburg, Salzburg, Austria Approved VIEWS 0 https://doi.org/10.5256/f1000research.13040.r24020 In the revised version Courtial et al. have addressed my concerns and ... Continue reading READ ALL In the revised version Courtial et al. have addressed my concerns and considered my suggestions in sufficient detail. I therefore recommend the manuscript for indexing. Competing Interests: No competing interests were disclosed. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Pitzschke A. Reviewer Report For: Validation of commercial ERK antibodies against the ERK orthologue of the scleractinian coral Stylophora pistillata [version 2; peer review: 3 approved] . F1000Research 2017, 6 :577 ( https://doi.org/10.5256/f1000research.13040.r24020 ) The direct URL for this report is: https://f1000research.com/articles/6-577/v2#referee-response-24020 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Respond or Comment COMMENT ON THIS REPORT Version 1 VERSION 1 PUBLISHED 26 Apr 2017 Views 0 Cite How to cite this report: Parages ML. Reviewer Report For: Validation of commercial ERK antibodies against the ERK orthologue of the scleractinian coral Stylophora pistillata [version 2; peer review: 3 approved] . F1000Research 2017, 6 :577 ( https://doi.org/10.5256/f1000research.12269.r23149 ) The direct URL for this report is: https://f1000research.com/articles/6-577/v1#referee-response-23149 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 22 Jun 2017 María L. Parages , Department of Ecology, Faculty of Sciences, University of Málaga, Málaga, Spain Approved with Reservations VIEWS 0 https://doi.org/10.5256/f1000research.12269.r23149 In this manuscript the authors Courtial et al. validate the use of commercial ERK antibodies for the detection of MAPK-like proteins in Coral. Although the paper is well written and has the quality to be indexed, a few issues should ... Continue reading READ ALL In this manuscript the authors Courtial et al. validate the use of commercial ERK antibodies for the detection of MAPK-like proteins in Coral. Although the paper is well written and has the quality to be indexed, a few issues should be figure out before its final acceptance. In terms of samples preparation and to allow future replication by others researchers would be appropriate to give more information about how to prepare the samples. For example, “Briefly, coral tissue was removed from the skeleton in 1 mL Laemmli buffer” so, how much coral tissue will be dissolved and resupended it in 1 mL of Laemmli Buffer? Less than 0.5 gr? More? While it is true that the authors note that immune-detection were performed as in Courtial et al. (2017), and that DTT (Dithiothreitol) and BFB (Bromophenol blue) were added to the samples and heated (5 minutes at 95°) before loading the gels, it is not specified how tissue extraction was performed. I must assume that the Lysis Buffer used was Laemmli Buffer? And in this case, how they have been unable to detect phosphorylated ERK? As far as I know lysis buffer for phosphorylated proteins usually contents EDTA or EGTA to chelate Mg2+/Ca2+, DTT for reduction of disulfide bonds, serine protease inhibitor (Aprotinin/Leupeptine), phosphatase inhibitors to block dephosphorylation like Na orthovanadate, or Beta-glycerophosphate (false substrate for phosphatase, between others… They also keep everything ice cold? Also, it surprises me that they used 3% low fat milk for membranes blocking, due to also screws up phosphor-tyrosine detection. Regarding the presence of an ERK ortholog in coral, the authors show the sequence with code Spi_isotig05348 (Spi_ERK) has the best hit with the human ERK protein (NP_0011035145) and they make reference to Liew et al., 2014. I was looking for this sequence into this paper, and I could not find it. The authors should provide in which database is the transcriptome, as well as, the Spi_ERK sequence itself. Is the work clearly and accurately presented and does it cite the current literature? Yes Are sufficient details of materials, methods and analysis provided to allow replication by others? Partly Are all the source data underlying the results available to ensure full reproducibility? Partly Are the conclusions drawn adequately supported by the results? Partly Competing Interests: No competing interests were disclosed. Reviewer Expertise: Molecular Ecology I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Parages ML. Reviewer Report For: Validation of commercial ERK antibodies against the ERK orthologue of the scleractinian coral Stylophora pistillata [version 2; peer review: 3 approved] . F1000Research 2017, 6 :577 ( https://doi.org/10.5256/f1000research.12269.r23149 ) The direct URL for this report is: https://f1000research.com/articles/6-577/v1#referee-response-23149 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Author Response 03 Jul 2017 Lucile Courtial , Marine Department, Centre Scientifique de Monaco, Monaco, MC-98000, Monaco 03 Jul 2017 Author Response In this manuscript the authors Courtial et al. validate the use of commercial ERK antibodies for the detection of MAPK-like proteins in Coral. Although the paper is well written and ... Continue reading In this manuscript the authors Courtial et al. validate the use of commercial ERK antibodies for the detection of MAPK-like proteins in Coral. Although the paper is well written and has the quality to be indexed, a few issues should be figure out before its final acceptance. We thank Dr. Parages for her comments and suggestions. To answer her concerns, we have added information and replaced a citation in the manuscript as detailed bellow. In terms of samples preparation and to allow future replication by others researchers would be appropriate to give more information about how to prepare the samples. For example, “Briefly, coral tissue was removed from the skeleton in 1 mL Laemmli buffer” so, how much coral tissue will be dissolved and resupended it in 1 mL of Laemmli Buffer? Less than 0.5 gr? More? All of the tissue from 3-5cm long S. pistillata nubbins was used. We added the precision in the materials and methods: P4: “Two small nubbins (3-5 cm long) were cut off from each mother colony and were allowed to heal for four weeks in 15 L open system tanks before the experiments.” P5: “Briefly, nubbins were airbrushed in 1 mL Laemmli buffer (2% SDS, 10% glycerol, 50mM Tris HCL pH7) ( Laemmli 1970) using an air-pick (5 bars) to remove the totality of the tissues surrounding the skeleton was removed from coral.” While it is true that the authors note that immune-detection were performed as in Courtial et al. (2017), and that DTT (Dithiothreitol) and BFB (Bromophenol blue) were added to the samples and heated (5 minutes at 95°) before loading the gels, it is not specified how tissue extraction was performed. I must assume that the Lysis Buffer used was Laemmli Buffer? And in this case, how they have been unable to detect phosphorylated ERK? As far as I know lysis buffer for phosphorylated proteins usually contents EDTA or EGTA to chelate Mg2+/Ca2+, DTT for reduction of disulfide bonds, serine protease inhibitor (Aprotinin/Leupeptine), phosphatase inhibitors to block dephosphorylation like Na orthovanadate, or Beta-glycerophosphate (false substrate for phosphatase, between others… Coral tissues were indeed lyzed in laemmli buffer. To prevent any doubt when reading the methods, we added a precision in the text: P3: “Briefly, nubbins were airbrushed in 1 mL Laemmli buffer (2% SDS, 10% glycerol, 50mM Tris HCL pH7) (Laemmli 1970) using an air-pick (5 bars) to remove the totality of the tissues surrounding the skeleton.” To answer the concern of Dr. Parages about the possible phosphatase activity in the samples, we would like to specify the fact that, due to a high concentration of SDS, the Laemmli buffer is a strongly denaturing buffer for proteins, including phosphatases. Therefore, this buffer, commonly used at room temperature, preserves phosphorylation of the proteins without the need to add phosphatases inhibitors (see Picco et al. 2016 for example). They also keep everything ice cold? Also, it surprises me that they used 3% low fat milk for membranes blocking, due to also screws up phosphor-tyrosine detection. We fully agree with Dr. Parages, the use of milk as a blocking buffer is usually not recommended for the detection of phospho-proteins as it may contain phospho-proteins that can interact with the anti-phospho primary antibodies. However, we have successfully used this blocking buffer in diverse experimental setups, including human cultured cells, ascidian embryos as well as coral lysates. We used this buffer in the course of this study because it gives far less background noise than any other blocking buffers tested. Regarding the presence of an ERK ortholog in coral, the authors show the sequence with code Spi_isotig05348 (Spi_ERK) has the best hit with the human ERK protein (NP_0011035145) and they make reference to Liew et al., 2014. I was looking for this sequence into this paper, and I could not find it. The authors should provide in which database is the transcriptome, as well as, the Spi_ERK sequence itself. We are grateful to the reviewer for questioning this particular point as it allowed us to uncover a significant error in the citation we used. The reference for the Spi EST containing the ERK orthologue open reading frame was obtained from the database generated during the study by Karako-Lampert, Zoccola et al. (Plos One 2014) and not the one by Liew et al (p.7) . The database containing the sequence can be downloaded from this address: http://data.centrescientifique.mc/CSMdata-stylodata.html . As mentioned in the manuscript, the ERK1 human protein sequence was blasted (tblastn) against the Karako-Lampert database using the blast tool hosted on a publically accessible local server of the Centre Scientifique de Monaco ( http://data.centrescientifique.mc/blast/blast.php ). The correct reference has been added to the manuscript. Due to current major security concerns for our servers, we have chosen not to include the aforementioned URL in the present manuscript. However, this URL is openly disclosed in the Karako-Lampert et al. paper, which should allow readers to access the database without the needing to contact corresponding authors. In this manuscript the authors Courtial et al. validate the use of commercial ERK antibodies for the detection of MAPK-like proteins in Coral. Although the paper is well written and has the quality to be indexed, a few issues should be figure out before its final acceptance. We thank Dr. Parages for her comments and suggestions. To answer her concerns, we have added information and replaced a citation in the manuscript as detailed bellow. In terms of samples preparation and to allow future replication by others researchers would be appropriate to give more information about how to prepare the samples. For example, “Briefly, coral tissue was removed from the skeleton in 1 mL Laemmli buffer” so, how much coral tissue will be dissolved and resupended it in 1 mL of Laemmli Buffer? Less than 0.5 gr? More? All of the tissue from 3-5cm long S. pistillata nubbins was used. We added the precision in the materials and methods: P4: “Two small nubbins (3-5 cm long) were cut off from each mother colony and were allowed to heal for four weeks in 15 L open system tanks before the experiments.” P5: “Briefly, nubbins were airbrushed in 1 mL Laemmli buffer (2% SDS, 10% glycerol, 50mM Tris HCL pH7) ( Laemmli 1970) using an air-pick (5 bars) to remove the totality of the tissues surrounding the skeleton was removed from coral.” While it is true that the authors note that immune-detection were performed as in Courtial et al. (2017), and that DTT (Dithiothreitol) and BFB (Bromophenol blue) were added to the samples and heated (5 minutes at 95°) before loading the gels, it is not specified how tissue extraction was performed. I must assume that the Lysis Buffer used was Laemmli Buffer? And in this case, how they have been unable to detect phosphorylated ERK? As far as I know lysis buffer for phosphorylated proteins usually contents EDTA or EGTA to chelate Mg2+/Ca2+, DTT for reduction of disulfide bonds, serine protease inhibitor (Aprotinin/Leupeptine), phosphatase inhibitors to block dephosphorylation like Na orthovanadate, or Beta-glycerophosphate (false substrate for phosphatase, between others… Coral tissues were indeed lyzed in laemmli buffer. To prevent any doubt when reading the methods, we added a precision in the text: P3: “Briefly, nubbins were airbrushed in 1 mL Laemmli buffer (2% SDS, 10% glycerol, 50mM Tris HCL pH7) (Laemmli 1970) using an air-pick (5 bars) to remove the totality of the tissues surrounding the skeleton.” To answer the concern of Dr. Parages about the possible phosphatase activity in the samples, we would like to specify the fact that, due to a high concentration of SDS, the Laemmli buffer is a strongly denaturing buffer for proteins, including phosphatases. Therefore, this buffer, commonly used at room temperature, preserves phosphorylation of the proteins without the need to add phosphatases inhibitors (see Picco et al. 2016 for example). They also keep everything ice cold? Also, it surprises me that they used 3% low fat milk for membranes blocking, due to also screws up phosphor-tyrosine detection. We fully agree with Dr. Parages, the use of milk as a blocking buffer is usually not recommended for the detection of phospho-proteins as it may contain phospho-proteins that can interact with the anti-phospho primary antibodies. However, we have successfully used this blocking buffer in diverse experimental setups, including human cultured cells, ascidian embryos as well as coral lysates. We used this buffer in the course of this study because it gives far less background noise than any other blocking buffers tested. Regarding the presence of an ERK ortholog in coral, the authors show the sequence with code Spi_isotig05348 (Spi_ERK) has the best hit with the human ERK protein (NP_0011035145) and they make reference to Liew et al., 2014. I was looking for this sequence into this paper, and I could not find it. The authors should provide in which database is the transcriptome, as well as, the Spi_ERK sequence itself. We are grateful to the reviewer for questioning this particular point as it allowed us to uncover a significant error in the citation we used. The reference for the Spi EST containing the ERK orthologue open reading frame was obtained from the database generated during the study by Karako-Lampert, Zoccola et al. (Plos One 2014) and not the one by Liew et al (p.7) . The database containing the sequence can be downloaded from this address: http://data.centrescientifique.mc/CSMdata-stylodata.html . As mentioned in the manuscript, the ERK1 human protein sequence was blasted (tblastn) against the Karako-Lampert database using the blast tool hosted on a publically accessible local server of the Centre Scientifique de Monaco ( http://data.centrescientifique.mc/blast/blast.php ). The correct reference has been added to the manuscript. Due to current major security concerns for our servers, we have chosen not to include the aforementioned URL in the present manuscript. However, this URL is openly disclosed in the Karako-Lampert et al. paper, which should allow readers to access the database without the needing to contact corresponding authors. Competing Interests: No competing interests were disclosed. Close Report a concern Respond or Comment COMMENTS ON THIS REPORT Author Response 03 Jul 2017 Lucile Courtial , Marine Department, Centre Scientifique de Monaco, Monaco, MC-98000, Monaco 03 Jul 2017 Author Response In this manuscript the authors Courtial et al. validate the use of commercial ERK antibodies for the detection of MAPK-like proteins in Coral. Although the paper is well written and ... Continue reading In this manuscript the authors Courtial et al. validate the use of commercial ERK antibodies for the detection of MAPK-like proteins in Coral. Although the paper is well written and has the quality to be indexed, a few issues should be figure out before its final acceptance. We thank Dr. Parages for her comments and suggestions. To answer her concerns, we have added information and replaced a citation in the manuscript as detailed bellow. In terms of samples preparation and to allow future replication by others researchers would be appropriate to give more information about how to prepare the samples. For example, “Briefly, coral tissue was removed from the skeleton in 1 mL Laemmli buffer” so, how much coral tissue will be dissolved and resupended it in 1 mL of Laemmli Buffer? Less than 0.5 gr? More? All of the tissue from 3-5cm long S. pistillata nubbins was used. We added the precision in the materials and methods: P4: “Two small nubbins (3-5 cm long) were cut off from each mother colony and were allowed to heal for four weeks in 15 L open system tanks before the experiments.” P5: “Briefly, nubbins were airbrushed in 1 mL Laemmli buffer (2% SDS, 10% glycerol, 50mM Tris HCL pH7) ( Laemmli 1970) using an air-pick (5 bars) to remove the totality of the tissues surrounding the skeleton was removed from coral.” While it is true that the authors note that immune-detection were performed as in Courtial et al. (2017), and that DTT (Dithiothreitol) and BFB (Bromophenol blue) were added to the samples and heated (5 minutes at 95°) before loading the gels, it is not specified how tissue extraction was performed. I must assume that the Lysis Buffer used was Laemmli Buffer? And in this case, how they have been unable to detect phosphorylated ERK? As far as I know lysis buffer for phosphorylated proteins usually contents EDTA or EGTA to chelate Mg2+/Ca2+, DTT for reduction of disulfide bonds, serine protease inhibitor (Aprotinin/Leupeptine), phosphatase inhibitors to block dephosphorylation like Na orthovanadate, or Beta-glycerophosphate (false substrate for phosphatase, between others… Coral tissues were indeed lyzed in laemmli buffer. To prevent any doubt when reading the methods, we added a precision in the text: P3: “Briefly, nubbins were airbrushed in 1 mL Laemmli buffer (2% SDS, 10% glycerol, 50mM Tris HCL pH7) (Laemmli 1970) using an air-pick (5 bars) to remove the totality of the tissues surrounding the skeleton.” To answer the concern of Dr. Parages about the possible phosphatase activity in the samples, we would like to specify the fact that, due to a high concentration of SDS, the Laemmli buffer is a strongly denaturing buffer for proteins, including phosphatases. Therefore, this buffer, commonly used at room temperature, preserves phosphorylation of the proteins without the need to add phosphatases inhibitors (see Picco et al. 2016 for example). They also keep everything ice cold? Also, it surprises me that they used 3% low fat milk for membranes blocking, due to also screws up phosphor-tyrosine detection. We fully agree with Dr. Parages, the use of milk as a blocking buffer is usually not recommended for the detection of phospho-proteins as it may contain phospho-proteins that can interact with the anti-phospho primary antibodies. However, we have successfully used this blocking buffer in diverse experimental setups, including human cultured cells, ascidian embryos as well as coral lysates. We used this buffer in the course of this study because it gives far less background noise than any other blocking buffers tested. Regarding the presence of an ERK ortholog in coral, the authors show the sequence with code Spi_isotig05348 (Spi_ERK) has the best hit with the human ERK protein (NP_0011035145) and they make reference to Liew et al., 2014. I was looking for this sequence into this paper, and I could not find it. The authors should provide in which database is the transcriptome, as well as, the Spi_ERK sequence itself. We are grateful to the reviewer for questioning this particular point as it allowed us to uncover a significant error in the citation we used. The reference for the Spi EST containing the ERK orthologue open reading frame was obtained from the database generated during the study by Karako-Lampert, Zoccola et al. (Plos One 2014) and not the one by Liew et al (p.7) . The database containing the sequence can be downloaded from this address: http://data.centrescientifique.mc/CSMdata-stylodata.html . As mentioned in the manuscript, the ERK1 human protein sequence was blasted (tblastn) against the Karako-Lampert database using the blast tool hosted on a publically accessible local server of the Centre Scientifique de Monaco ( http://data.centrescientifique.mc/blast/blast.php ). The correct reference has been added to the manuscript. Due to current major security concerns for our servers, we have chosen not to include the aforementioned URL in the present manuscript. However, this URL is openly disclosed in the Karako-Lampert et al. paper, which should allow readers to access the database without the needing to contact corresponding authors. In this manuscript the authors Courtial et al. validate the use of commercial ERK antibodies for the detection of MAPK-like proteins in Coral. Although the paper is well written and has the quality to be indexed, a few issues should be figure out before its final acceptance. We thank Dr. Parages for her comments and suggestions. To answer her concerns, we have added information and replaced a citation in the manuscript as detailed bellow. In terms of samples preparation and to allow future replication by others researchers would be appropriate to give more information about how to prepare the samples. For example, “Briefly, coral tissue was removed from the skeleton in 1 mL Laemmli buffer” so, how much coral tissue will be dissolved and resupended it in 1 mL of Laemmli Buffer? Less than 0.5 gr? More? All of the tissue from 3-5cm long S. pistillata nubbins was used. We added the precision in the materials and methods: P4: “Two small nubbins (3-5 cm long) were cut off from each mother colony and were allowed to heal for four weeks in 15 L open system tanks before the experiments.” P5: “Briefly, nubbins were airbrushed in 1 mL Laemmli buffer (2% SDS, 10% glycerol, 50mM Tris HCL pH7) ( Laemmli 1970) using an air-pick (5 bars) to remove the totality of the tissues surrounding the skeleton was removed from coral.” While it is true that the authors note that immune-detection were performed as in Courtial et al. (2017), and that DTT (Dithiothreitol) and BFB (Bromophenol blue) were added to the samples and heated (5 minutes at 95°) before loading the gels, it is not specified how tissue extraction was performed. I must assume that the Lysis Buffer used was Laemmli Buffer? And in this case, how they have been unable to detect phosphorylated ERK? As far as I know lysis buffer for phosphorylated proteins usually contents EDTA or EGTA to chelate Mg2+/Ca2+, DTT for reduction of disulfide bonds, serine protease inhibitor (Aprotinin/Leupeptine), phosphatase inhibitors to block dephosphorylation like Na orthovanadate, or Beta-glycerophosphate (false substrate for phosphatase, between others… Coral tissues were indeed lyzed in laemmli buffer. To prevent any doubt when reading the methods, we added a precision in the text: P3: “Briefly, nubbins were airbrushed in 1 mL Laemmli buffer (2% SDS, 10% glycerol, 50mM Tris HCL pH7) (Laemmli 1970) using an air-pick (5 bars) to remove the totality of the tissues surrounding the skeleton.” To answer the concern of Dr. Parages about the possible phosphatase activity in the samples, we would like to specify the fact that, due to a high concentration of SDS, the Laemmli buffer is a strongly denaturing buffer for proteins, including phosphatases. Therefore, this buffer, commonly used at room temperature, preserves phosphorylation of the proteins without the need to add phosphatases inhibitors (see Picco et al. 2016 for example). They also keep everything ice cold? Also, it surprises me that they used 3% low fat milk for membranes blocking, due to also screws up phosphor-tyrosine detection. We fully agree with Dr. Parages, the use of milk as a blocking buffer is usually not recommended for the detection of phospho-proteins as it may contain phospho-proteins that can interact with the anti-phospho primary antibodies. However, we have successfully used this blocking buffer in diverse experimental setups, including human cultured cells, ascidian embryos as well as coral lysates. We used this buffer in the course of this study because it gives far less background noise than any other blocking buffers tested. Regarding the presence of an ERK ortholog in coral, the authors show the sequence with code Spi_isotig05348 (Spi_ERK) has the best hit with the human ERK protein (NP_0011035145) and they make reference to Liew et al., 2014. I was looking for this sequence into this paper, and I could not find it. The authors should provide in which database is the transcriptome, as well as, the Spi_ERK sequence itself. We are grateful to the reviewer for questioning this particular point as it allowed us to uncover a significant error in the citation we used. The reference for the Spi EST containing the ERK orthologue open reading frame was obtained from the database generated during the study by Karako-Lampert, Zoccola et al. (Plos One 2014) and not the one by Liew et al (p.7) . The database containing the sequence can be downloaded from this address: http://data.centrescientifique.mc/CSMdata-stylodata.html . As mentioned in the manuscript, the ERK1 human protein sequence was blasted (tblastn) against the Karako-Lampert database using the blast tool hosted on a publically accessible local server of the Centre Scientifique de Monaco ( http://data.centrescientifique.mc/blast/blast.php ). The correct reference has been added to the manuscript. Due to current major security concerns for our servers, we have chosen not to include the aforementioned URL in the present manuscript. However, this URL is openly disclosed in the Karako-Lampert et al. paper, which should allow readers to access the database without the needing to contact corresponding authors. Competing Interests: No competing interests were disclosed. Close Report a concern COMMENT ON THIS REPORT Views 0 Cite How to cite this report: Castellano I. Reviewer Report For: Validation of commercial ERK antibodies against the ERK orthologue of the scleractinian coral Stylophora pistillata [version 2; peer review: 3 approved] . F1000Research 2017, 6 :577 ( https://doi.org/10.5256/f1000research.12269.r23532 ) The direct URL for this report is: https://f1000research.com/articles/6-577/v1#referee-response-23532 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 16 Jun 2017 Immacolata Castellano , Department of Biology and Evolution of Marine Organisms, Stazione Zoologica Anton Dohrn, Naples, Italy Approved VIEWS 0 https://doi.org/10.5256/f1000research.12269.r23532 The paper by Courtial et al. describes the cross-reactivity of two commercial antibodies produced against the mammalian forms of ERK for the scleractinian coral Stylophora pistillata. This should open new perspective for the study of ERK signalling in response to ... Continue reading READ ALL The paper by Courtial et al. describes the cross-reactivity of two commercial antibodies produced against the mammalian forms of ERK for the scleractinian coral Stylophora pistillata. This should open new perspective for the study of ERK signalling in response to different environmental cues. The paper is clear and well written, however it lacks of some references. In the Introduction, the authors should cite other invertebrates where ERK signalling is known to be conserved and regulated by environmental cues, for example Ciona intestinalis (Castellano et al, PlOS One 2014, Castellano et al, Open Biology 2015). Similarly, in Results and Discussion, when the authors say that .. "a single orthologue of ERK is present in these cnidarians, as opposed to the two genes encoding ERKs in most mammalian genomes”, they should specify that also in other invertebrates, only one ERK form was found (Russo et al, JBC 2004; Castellano et al, PloS One 2014). Also the use of the MEK inhibitor U0126 was assessed in C. intestinalis (Castellano et al, Open Biology 2015). Finally the authors pay attention along the text to some errors, i.e. change “Thr2020/204” with Thr202/204, and “through this medium” with “through this method”. Is the work clearly and accurately presented and does it cite the current literature? Partly Are sufficient details of materials, methods and analysis provided to allow replication by others? Yes Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes Competing Interests: No competing interests were disclosed. Reviewer Expertise: Biochemistry I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Castellano I. Reviewer Report For: Validation of commercial ERK antibodies against the ERK orthologue of the scleractinian coral Stylophora pistillata [version 2; peer review: 3 approved] . F1000Research 2017, 6 :577 ( https://doi.org/10.5256/f1000research.12269.r23532 ) The direct URL for this report is: https://f1000research.com/articles/6-577/v1#referee-response-23532 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Author Response 03 Jul 2017 Lucile Courtial , Marine Department, Centre Scientifique de Monaco, Monaco, MC-98000, Monaco 03 Jul 2017 Author Response The paper by Courtial et al. describes the cross-reactivity of two commercial antibodies produced against the mammalian forms of ERK for the scleractinian coral Stylophora pistillata. This should open new ... Continue reading The paper by Courtial et al. describes the cross-reactivity of two commercial antibodies produced against the mammalian forms of ERK for the scleractinian coral Stylophora pistillata. This should open new perspective for the study of ERK signalling in response to different environmental cues. The paper is clear and well written, however it lacks of some references. We thank Dr. Castellano for her comments and suggestions. We have added information to the manuscript to answer her concerns as detailed bellow. In the Introduction, the authors should cite other invertebrates where ERK signalling is known to be conserved and regulated by environmental cues, for example Ciona intestinalis (Castellano et al, PlOS One 2014, Castellano et al, Open Biology 2015). We added a precision and the reference in the text: “The ERK gene family is evolutionnarily conserved and is found in all eukaryotes, including yeasts, plants, vertebrates and invertebrates (Widmann et al. 1999; Chen et al. 2001; Castellano et al. 2014).” Similarly, in Results and Discussion, when the authors say that .. "a single orthologue of ERK is present in these cnidarians, as opposed to the two genes encoding ERKs in most mammalian genomes”, they should specify that also in other invertebrates, only one ERK form was found (Russo et al, JBC 2004; Castellano et al, PloS One 2014). We added the precision in the text:P4 “This result suggests that a single ortholog of ERK is present in these cnidarians, consistently with previous work where only one ERK ortholog was found (Russo et al. 2004; Castellano et al. 2014) but as opposed to the two genes encoding ERKs in most mammalian genomes (Ip and Davis 1998).” Also the use of the MEK inhibitor U0126 was assessed in C. intestinalis (Castellano et al, Open Biology 2015). Despite extensive search into the reference cited by Dr. Castellano, we could not find any experiment using U0126 in this paper. The aforementioned reference instead reports the use of a dual specificity phosphatase inhibitor. Moreover, the work of Picco et al. (2007) cited in the manuscript already reports the use of U0126 in Ciona embryos. We therefore did not include the suggested citation in the text. Finally the authors pay attention along the text to some errors, i.e. change “Thr2020/204” with Thr202/204, and “through this medium” with “through this method”. We changed the errors in the revised manuscript. The paper by Courtial et al. describes the cross-reactivity of two commercial antibodies produced against the mammalian forms of ERK for the scleractinian coral Stylophora pistillata. This should open new perspective for the study of ERK signalling in response to different environmental cues. The paper is clear and well written, however it lacks of some references. We thank Dr. Castellano for her comments and suggestions. We have added information to the manuscript to answer her concerns as detailed bellow. In the Introduction, the authors should cite other invertebrates where ERK signalling is known to be conserved and regulated by environmental cues, for example Ciona intestinalis (Castellano et al, PlOS One 2014, Castellano et al, Open Biology 2015). We added a precision and the reference in the text: “The ERK gene family is evolutionnarily conserved and is found in all eukaryotes, including yeasts, plants, vertebrates and invertebrates (Widmann et al. 1999; Chen et al. 2001; Castellano et al. 2014).” Similarly, in Results and Discussion, when the authors say that .. "a single orthologue of ERK is present in these cnidarians, as opposed to the two genes encoding ERKs in most mammalian genomes”, they should specify that also in other invertebrates, only one ERK form was found (Russo et al, JBC 2004; Castellano et al, PloS One 2014). We added the precision in the text:P4 “This result suggests that a single ortholog of ERK is present in these cnidarians, consistently with previous work where only one ERK ortholog was found (Russo et al. 2004; Castellano et al. 2014) but as opposed to the two genes encoding ERKs in most mammalian genomes (Ip and Davis 1998).” Also the use of the MEK inhibitor U0126 was assessed in C. intestinalis (Castellano et al, Open Biology 2015). Despite extensive search into the reference cited by Dr. Castellano, we could not find any experiment using U0126 in this paper. The aforementioned reference instead reports the use of a dual specificity phosphatase inhibitor. Moreover, the work of Picco et al. (2007) cited in the manuscript already reports the use of U0126 in Ciona embryos. We therefore did not include the suggested citation in the text. Finally the authors pay attention along the text to some errors, i.e. change “Thr2020/204” with Thr202/204, and “through this medium” with “through this method”. We changed the errors in the revised manuscript. Competing Interests: No competing interests were disclosed. Close Report a concern Respond or Comment COMMENTS ON THIS REPORT Author Response 03 Jul 2017 Lucile Courtial , Marine Department, Centre Scientifique de Monaco, Monaco, MC-98000, Monaco 03 Jul 2017 Author Response The paper by Courtial et al. describes the cross-reactivity of two commercial antibodies produced against the mammalian forms of ERK for the scleractinian coral Stylophora pistillata. This should open new ... Continue reading The paper by Courtial et al. describes the cross-reactivity of two commercial antibodies produced against the mammalian forms of ERK for the scleractinian coral Stylophora pistillata. This should open new perspective for the study of ERK signalling in response to different environmental cues. The paper is clear and well written, however it lacks of some references. We thank Dr. Castellano for her comments and suggestions. We have added information to the manuscript to answer her concerns as detailed bellow. In the Introduction, the authors should cite other invertebrates where ERK signalling is known to be conserved and regulated by environmental cues, for example Ciona intestinalis (Castellano et al, PlOS One 2014, Castellano et al, Open Biology 2015). We added a precision and the reference in the text: “The ERK gene family is evolutionnarily conserved and is found in all eukaryotes, including yeasts, plants, vertebrates and invertebrates (Widmann et al. 1999; Chen et al. 2001; Castellano et al. 2014).” Similarly, in Results and Discussion, when the authors say that .. "a single orthologue of ERK is present in these cnidarians, as opposed to the two genes encoding ERKs in most mammalian genomes”, they should specify that also in other invertebrates, only one ERK form was found (Russo et al, JBC 2004; Castellano et al, PloS One 2014). We added the precision in the text:P4 “This result suggests that a single ortholog of ERK is present in these cnidarians, consistently with previous work where only one ERK ortholog was found (Russo et al. 2004; Castellano et al. 2014) but as opposed to the two genes encoding ERKs in most mammalian genomes (Ip and Davis 1998).” Also the use of the MEK inhibitor U0126 was assessed in C. intestinalis (Castellano et al, Open Biology 2015). Despite extensive search into the reference cited by Dr. Castellano, we could not find any experiment using U0126 in this paper. The aforementioned reference instead reports the use of a dual specificity phosphatase inhibitor. Moreover, the work of Picco et al. (2007) cited in the manuscript already reports the use of U0126 in Ciona embryos. We therefore did not include the suggested citation in the text. Finally the authors pay attention along the text to some errors, i.e. change “Thr2020/204” with Thr202/204, and “through this medium” with “through this method”. We changed the errors in the revised manuscript. The paper by Courtial et al. describes the cross-reactivity of two commercial antibodies produced against the mammalian forms of ERK for the scleractinian coral Stylophora pistillata. This should open new perspective for the study of ERK signalling in response to different environmental cues. The paper is clear and well written, however it lacks of some references. We thank Dr. Castellano for her comments and suggestions. We have added information to the manuscript to answer her concerns as detailed bellow. In the Introduction, the authors should cite other invertebrates where ERK signalling is known to be conserved and regulated by environmental cues, for example Ciona intestinalis (Castellano et al, PlOS One 2014, Castellano et al, Open Biology 2015). We added a precision and the reference in the text: “The ERK gene family is evolutionnarily conserved and is found in all eukaryotes, including yeasts, plants, vertebrates and invertebrates (Widmann et al. 1999; Chen et al. 2001; Castellano et al. 2014).” Similarly, in Results and Discussion, when the authors say that .. "a single orthologue of ERK is present in these cnidarians, as opposed to the two genes encoding ERKs in most mammalian genomes”, they should specify that also in other invertebrates, only one ERK form was found (Russo et al, JBC 2004; Castellano et al, PloS One 2014). We added the precision in the text:P4 “This result suggests that a single ortholog of ERK is present in these cnidarians, consistently with previous work where only one ERK ortholog was found (Russo et al. 2004; Castellano et al. 2014) but as opposed to the two genes encoding ERKs in most mammalian genomes (Ip and Davis 1998).” Also the use of the MEK inhibitor U0126 was assessed in C. intestinalis (Castellano et al, Open Biology 2015). Despite extensive search into the reference cited by Dr. Castellano, we could not find any experiment using U0126 in this paper. The aforementioned reference instead reports the use of a dual specificity phosphatase inhibitor. Moreover, the work of Picco et al. (2007) cited in the manuscript already reports the use of U0126 in Ciona embryos. We therefore did not include the suggested citation in the text. Finally the authors pay attention along the text to some errors, i.e. change “Thr2020/204” with Thr202/204, and “through this medium” with “through this method”. We changed the errors in the revised manuscript. Competing Interests: No competing interests were disclosed. Close Report a concern COMMENT ON THIS REPORT Views 0 Cite How to cite this report: Pitzschke A. Reviewer Report For: Validation of commercial ERK antibodies against the ERK orthologue of the scleractinian coral Stylophora pistillata [version 2; peer review: 3 approved] . F1000Research 2017, 6 :577 ( https://doi.org/10.5256/f1000research.12269.r22707 ) The direct URL for this report is: https://f1000research.com/articles/6-577/v1#referee-response-22707 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 30 May 2017 Andrea Pitzschke , Department of Cell Biology and Physiology, University of Salzburg, Salzburg, Austria Approved with Reservations VIEWS 0 https://doi.org/10.5256/f1000research.12269.r22707 The manuscript provides first insight into a putative MAPK in the coral Stylophora pistillata. Experiments include protein extraction and immunoblot analysis. Overall: The experiments that had been performed are properly designed. However, the manuscript ... Continue reading READ ALL The manuscript provides first insight into a putative MAPK in the coral Stylophora pistillata. Experiments include protein extraction and immunoblot analysis. Overall: The experiments that had been performed are properly designed. However, the manuscript lacks sufficiently-detailed information, as well as controls (protein loading). Conclusions are premature or should be re-phrased. Detailed points of criticism: Title “orthologue” is inappropriate. Should be “homologue”. Methods P3 “small rubbins selected” – please be more specific about size and sampling: “tissue removed from coral”: be more specific. Tissue primarily from the surface, how deep was the cut into the material? (I suggest to include a schematic figure incl. scale-bar). This information is important because inhibitors (e.g. UO126) will only diffuse over a short distance, i.e. not reach deeper layers. P4: “extensive washing”: duration and number of solution changes missing Fig.2B: “% or control” rather OF control. The error bar in the control sample is irrelevant, as it is defined as strictly 100%. There is no documentation of protein loading (e.g. Coomassie-stained membrane after immunodetection). The U126-independent intensity of the ERK-Signal is insufficient as control. Conclusions: “…antibody can be used…in different experimental or environmental conditions” This conclusion is premature. As a minimum, the authors should perform an induction experiment. The inhibitory approach (U126) only evidences that a MAPKK is the upstream regulator. Coral research community will only benefit from the antibody and the current study if dynamic ERK activity responses can be monitored. Is the work clearly and accurately presented and does it cite the current literature? Partly Are sufficient details of materials, methods and analysis provided to allow replication by others? Partly Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Partly Competing Interests: No competing interests were disclosed. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Pitzschke A. Reviewer Report For: Validation of commercial ERK antibodies against the ERK orthologue of the scleractinian coral Stylophora pistillata [version 2; peer review: 3 approved] . F1000Research 2017, 6 :577 ( https://doi.org/10.5256/f1000research.12269.r22707 ) The direct URL for this report is: https://f1000research.com/articles/6-577/v1#referee-response-22707 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Author Response 03 Jul 2017 Lucile Courtial , Marine Department, Centre Scientifique de Monaco, Monaco, MC-98000, Monaco 03 Jul 2017 Author Response Overall: The experiments that had been performed are properly designed. However, the manuscript lacks sufficiently-detailed information, as well as controls (protein loading). Conclusions are premature or should be re-phrased. ... Continue reading Overall: The experiments that had been performed are properly designed. However, the manuscript lacks sufficiently-detailed information, as well as controls (protein loading). Conclusions are premature or should be re-phrased. We thank Dr. Pitzschke for her comments and suggestions. We have added information to the manuscript and performed an additional experiment to answer her concerns as detailed bellow. Detailed points of criticism: Title “orthologue” is inappropriate. Should be “homologue”. We have used the term “orthologue” in our manuscript in its definition notably given by Walter Fitch (Fitch 1970, 2000), that is: “Orthology is that relationship where sequence divergence follows speciation, that is, where the common ancestor of the two genes lies in the common ancestor of the taxa from which the two sequences were obtained”. Dr. Pitzschke is right, the Spi- and Hs-ERK proteins are homologues but, on top of that, they also are orthologues. We therefore still think that the term “orthologue” is more accurate in our case. Methods P3 “small nubbins selected” –please be more specific about size and sampling: “tissue removed from coral”: be more specific. Tissue primarily from the surface, how deep was the cut into the material? To be more specific, we added the following sentences in the revised manuscript: P2: “Two small nubbins (3-5 cm long) were cut off from each mother colony and were allowed to heal for four weeks in 15 L open system tanks before the experiments.” P3: “Briefly, nubbins were airbrushed in 1 mL Laemmli buffer ( i.e. lysing buffer, 1.5 X, Laemmli 1970) using an air-pick (5 bars) to remove the totality of the tissues surrounding the skeleton was removed from coral.” This information is important because inhibitors (e.g. UO126) will only diffuse over a short distance, i.e. not reach deeper layers. We added a sentence to prevent further doubts concerning the bioavailability of U0126. P6: “In order to test the specificity of the antibodies, we therefore used U0126, a very potent and selective inhibitor of MEK (Bain et al. 2007). The limited thickness of the animal tissue covering the skeleton and the very large surface of contact of both ectoderm and endoderm with the seawater render S. pistillata suitable for treatment with drugs directly diluted in the seawater as we previously showed (Courtial et al. 2017) …” P4: “extensive washing”: duration and number of solution changes missing We added precisions in the materials and methods: P4 : “4 x 30 minutes” Fig.2B: “% or control” rather OF control. The error bar in the control sample is irrelevant, as it is defined as strictly 100%. There is no documentation of protein loading (e.g. Coomassie-stained membrane after immunodetection). The U126-independent intensity of the ERK-Signal is insufficient as control. We added the amido black colored membranes in Figure 2 and Figure S1 as a loading control. Conclusions: “…antibody can be used…in different experimental or environmental conditions” This conclusion is premature. As a minimum, the authors should perform an induction experiment. The inhibitory approach (U126) only evidences that a MAPKK is the upstream regulator. Coral research community will only benefit from the antibody and the current study if dynamic ERK activity responses can be monitored. We performed an additional experiment and added a figure and related text in the manuscript to justify our statement (P9). Overall: The experiments that had been performed are properly designed. However, the manuscript lacks sufficiently-detailed information, as well as controls (protein loading). Conclusions are premature or should be re-phrased. We thank Dr. Pitzschke for her comments and suggestions. We have added information to the manuscript and performed an additional experiment to answer her concerns as detailed bellow. Detailed points of criticism: Title “orthologue” is inappropriate. Should be “homologue”. We have used the term “orthologue” in our manuscript in its definition notably given by Walter Fitch (Fitch 1970, 2000), that is: “Orthology is that relationship where sequence divergence follows speciation, that is, where the common ancestor of the two genes lies in the common ancestor of the taxa from which the two sequences were obtained”. Dr. Pitzschke is right, the Spi- and Hs-ERK proteins are homologues but, on top of that, they also are orthologues. We therefore still think that the term “orthologue” is more accurate in our case. Methods P3 “small nubbins selected” –please be more specific about size and sampling: “tissue removed from coral”: be more specific. Tissue primarily from the surface, how deep was the cut into the material? To be more specific, we added the following sentences in the revised manuscript: P2: “Two small nubbins (3-5 cm long) were cut off from each mother colony and were allowed to heal for four weeks in 15 L open system tanks before the experiments.” P3: “Briefly, nubbins were airbrushed in 1 mL Laemmli buffer ( i.e. lysing buffer, 1.5 X, Laemmli 1970) using an air-pick (5 bars) to remove the totality of the tissues surrounding the skeleton was removed from coral.” This information is important because inhibitors (e.g. UO126) will only diffuse over a short distance, i.e. not reach deeper layers. We added a sentence to prevent further doubts concerning the bioavailability of U0126. P6: “In order to test the specificity of the antibodies, we therefore used U0126, a very potent and selective inhibitor of MEK (Bain et al. 2007). The limited thickness of the animal tissue covering the skeleton and the very large surface of contact of both ectoderm and endoderm with the seawater render S. pistillata suitable for treatment with drugs directly diluted in the seawater as we previously showed (Courtial et al. 2017) …” P4: “extensive washing”: duration and number of solution changes missing We added precisions in the materials and methods: P4 : “4 x 30 minutes” Fig.2B: “% or control” rather OF control. The error bar in the control sample is irrelevant, as it is defined as strictly 100%. There is no documentation of protein loading (e.g. Coomassie-stained membrane after immunodetection). The U126-independent intensity of the ERK-Signal is insufficient as control. We added the amido black colored membranes in Figure 2 and Figure S1 as a loading control. Conclusions: “…antibody can be used…in different experimental or environmental conditions” This conclusion is premature. As a minimum, the authors should perform an induction experiment. The inhibitory approach (U126) only evidences that a MAPKK is the upstream regulator. Coral research community will only benefit from the antibody and the current study if dynamic ERK activity responses can be monitored. We performed an additional experiment and added a figure and related text in the manuscript to justify our statement (P9). Competing Interests: No competing interests were disclosed. Close Report a concern Respond or Comment COMMENTS ON THIS REPORT Author Response 03 Jul 2017 Lucile Courtial , Marine Department, Centre Scientifique de Monaco, Monaco, MC-98000, Monaco 03 Jul 2017 Author Response Overall: The experiments that had been performed are properly designed. However, the manuscript lacks sufficiently-detailed information, as well as controls (protein loading). Conclusions are premature or should be re-phrased. ... Continue reading Overall: The experiments that had been performed are properly designed. However, the manuscript lacks sufficiently-detailed information, as well as controls (protein loading). Conclusions are premature or should be re-phrased. We thank Dr. Pitzschke for her comments and suggestions. We have added information to the manuscript and performed an additional experiment to answer her concerns as detailed bellow. Detailed points of criticism: Title “orthologue” is inappropriate. Should be “homologue”. We have used the term “orthologue” in our manuscript in its definition notably given by Walter Fitch (Fitch 1970, 2000), that is: “Orthology is that relationship where sequence divergence follows speciation, that is, where the common ancestor of the two genes lies in the common ancestor of the taxa from which the two sequences were obtained”. Dr. Pitzschke is right, the Spi- and Hs-ERK proteins are homologues but, on top of that, they also are orthologues. We therefore still think that the term “orthologue” is more accurate in our case. Methods P3 “small nubbins selected” –please be more specific about size and sampling: “tissue removed from coral”: be more specific. Tissue primarily from the surface, how deep was the cut into the material? To be more specific, we added the following sentences in the revised manuscript: P2: “Two small nubbins (3-5 cm long) were cut off from each mother colony and were allowed to heal for four weeks in 15 L open system tanks before the experiments.” P3: “Briefly, nubbins were airbrushed in 1 mL Laemmli buffer ( i.e. lysing buffer, 1.5 X, Laemmli 1970) using an air-pick (5 bars) to remove the totality of the tissues surrounding the skeleton was removed from coral.” This information is important because inhibitors (e.g. UO126) will only diffuse over a short distance, i.e. not reach deeper layers. We added a sentence to prevent further doubts concerning the bioavailability of U0126. P6: “In order to test the specificity of the antibodies, we therefore used U0126, a very potent and selective inhibitor of MEK (Bain et al. 2007). The limited thickness of the animal tissue covering the skeleton and the very large surface of contact of both ectoderm and endoderm with the seawater render S. pistillata suitable for treatment with drugs directly diluted in the seawater as we previously showed (Courtial et al. 2017) …” P4: “extensive washing”: duration and number of solution changes missing We added precisions in the materials and methods: P4 : “4 x 30 minutes” Fig.2B: “% or control” rather OF control. The error bar in the control sample is irrelevant, as it is defined as strictly 100%. There is no documentation of protein loading (e.g. Coomassie-stained membrane after immunodetection). The U126-independent intensity of the ERK-Signal is insufficient as control. We added the amido black colored membranes in Figure 2 and Figure S1 as a loading control. Conclusions: “…antibody can be used…in different experimental or environmental conditions” This conclusion is premature. As a minimum, the authors should perform an induction experiment. The inhibitory approach (U126) only evidences that a MAPKK is the upstream regulator. Coral research community will only benefit from the antibody and the current study if dynamic ERK activity responses can be monitored. We performed an additional experiment and added a figure and related text in the manuscript to justify our statement (P9). Overall: The experiments that had been performed are properly designed. However, the manuscript lacks sufficiently-detailed information, as well as controls (protein loading). Conclusions are premature or should be re-phrased. We thank Dr. Pitzschke for her comments and suggestions. We have added information to the manuscript and performed an additional experiment to answer her concerns as detailed bellow. Detailed points of criticism: Title “orthologue” is inappropriate. Should be “homologue”. We have used the term “orthologue” in our manuscript in its definition notably given by Walter Fitch (Fitch 1970, 2000), that is: “Orthology is that relationship where sequence divergence follows speciation, that is, where the common ancestor of the two genes lies in the common ancestor of the taxa from which the two sequences were obtained”. Dr. Pitzschke is right, the Spi- and Hs-ERK proteins are homologues but, on top of that, they also are orthologues. We therefore still think that the term “orthologue” is more accurate in our case. Methods P3 “small nubbins selected” –please be more specific about size and sampling: “tissue removed from coral”: be more specific. Tissue primarily from the surface, how deep was the cut into the material? To be more specific, we added the following sentences in the revised manuscript: P2: “Two small nubbins (3-5 cm long) were cut off from each mother colony and were allowed to heal for four weeks in 15 L open system tanks before the experiments.” P3: “Briefly, nubbins were airbrushed in 1 mL Laemmli buffer ( i.e. lysing buffer, 1.5 X, Laemmli 1970) using an air-pick (5 bars) to remove the totality of the tissues surrounding the skeleton was removed from coral.” This information is important because inhibitors (e.g. UO126) will only diffuse over a short distance, i.e. not reach deeper layers. We added a sentence to prevent further doubts concerning the bioavailability of U0126. P6: “In order to test the specificity of the antibodies, we therefore used U0126, a very potent and selective inhibitor of MEK (Bain et al. 2007). The limited thickness of the animal tissue covering the skeleton and the very large surface of contact of both ectoderm and endoderm with the seawater render S. pistillata suitable for treatment with drugs directly diluted in the seawater as we previously showed (Courtial et al. 2017) …” P4: “extensive washing”: duration and number of solution changes missing We added precisions in the materials and methods: P4 : “4 x 30 minutes” Fig.2B: “% or control” rather OF control. The error bar in the control sample is irrelevant, as it is defined as strictly 100%. There is no documentation of protein loading (e.g. Coomassie-stained membrane after immunodetection). The U126-independent intensity of the ERK-Signal is insufficient as control. We added the amido black colored membranes in Figure 2 and Figure S1 as a loading control. Conclusions: “…antibody can be used…in different experimental or environmental conditions” This conclusion is premature. As a minimum, the authors should perform an induction experiment. The inhibitory approach (U126) only evidences that a MAPKK is the upstream regulator. Coral research community will only benefit from the antibody and the current study if dynamic ERK activity responses can be monitored. We performed an additional experiment and added a figure and related text in the manuscript to justify our statement (P9). Competing Interests: No competing interests were disclosed. Close Report a concern COMMENT ON THIS REPORT Comments on this article Comments (0) Version 2 VERSION 2 PUBLISHED 26 Apr 2017 ADD YOUR COMMENT Comment keyboard_arrow_left keyboard_arrow_right Open Peer Review Reviewer Status info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Reviewer Reports Invited Reviewers 1 2 3 Version 2 (revision) 03 Jul 17 read read Version 1 26 Apr 17 read read read Andrea Pitzschke , University of Salzburg, Salzburg, Austria Immacolata Castellano , Stazione Zoologica Anton Dohrn, Naples, Italy María L. Parages , University of Málaga, Málaga, Spain Comments on this article All Comments (0) Add a comment Sign up for content alerts Sign Up You are now signed up to receive this alert Browse by related subjects keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2017 Parages M. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 21 Jul 2017 | for Version 2 María L. Parages , Department of Ecology, Faculty of Sciences, University of Málaga, Málaga, Spain 0 Views copyright © 2017 Parages M. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions After author response, I recommend the manuscript for indexing. So I suggest the new status as Approved. Competing Interests No competing interests were disclosed. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. reply Respond to this report Responses (0) Parages ML. Peer Review Report For: Validation of commercial ERK antibodies against the ERK orthologue of the scleractinian coral Stylophora pistillata [version 2; peer review: 3 approved] . F1000Research 2017, 6 :577 ( https://doi.org/10.5256/f1000research.13040.r23990) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/6-577/v2#referee-response-23990 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2017 Pitzschke A. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 04 Jul 2017 | for Version 2 Andrea Pitzschke , Department of Cell Biology and Physiology, University of Salzburg, Salzburg, Austria 0 Views copyright © 2017 Pitzschke A. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions In the revised version Courtial et al. have addressed my concerns and considered my suggestions in sufficient detail. I therefore recommend the manuscript for indexing. Competing Interests No competing interests were disclosed. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. reply Respond to this report Responses (0) Pitzschke A. Peer Review Report For: Validation of commercial ERK antibodies against the ERK orthologue of the scleractinian coral Stylophora pistillata [version 2; peer review: 3 approved] . F1000Research 2017, 6 :577 ( https://doi.org/10.5256/f1000research.13040.r24020) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/6-577/v2#referee-response-24020 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2017 Parages M. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 22 Jun 2017 | for Version 1 María L. Parages , Department of Ecology, Faculty of Sciences, University of Málaga, Málaga, Spain 0 Views copyright © 2017 Parages M. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (1) Approved With Reservations info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions In this manuscript the authors Courtial et al. validate the use of commercial ERK antibodies for the detection of MAPK-like proteins in Coral. Although the paper is well written and has the quality to be indexed, a few issues should be figure out before its final acceptance. In terms of samples preparation and to allow future replication by others researchers would be appropriate to give more information about how to prepare the samples. For example, “Briefly, coral tissue was removed from the skeleton in 1 mL Laemmli buffer” so, how much coral tissue will be dissolved and resupended it in 1 mL of Laemmli Buffer? Less than 0.5 gr? More? While it is true that the authors note that immune-detection were performed as in Courtial et al. (2017), and that DTT (Dithiothreitol) and BFB (Bromophenol blue) were added to the samples and heated (5 minutes at 95°) before loading the gels, it is not specified how tissue extraction was performed. I must assume that the Lysis Buffer used was Laemmli Buffer? And in this case, how they have been unable to detect phosphorylated ERK? As far as I know lysis buffer for phosphorylated proteins usually contents EDTA or EGTA to chelate Mg2+/Ca2+, DTT for reduction of disulfide bonds, serine protease inhibitor (Aprotinin/Leupeptine), phosphatase inhibitors to block dephosphorylation like Na orthovanadate, or Beta-glycerophosphate (false substrate for phosphatase, between others… They also keep everything ice cold? Also, it surprises me that they used 3% low fat milk for membranes blocking, due to also screws up phosphor-tyrosine detection. Regarding the presence of an ERK ortholog in coral, the authors show the sequence with code Spi_isotig05348 (Spi_ERK) has the best hit with the human ERK protein (NP_0011035145) and they make reference to Liew et al., 2014. I was looking for this sequence into this paper, and I could not find it. The authors should provide in which database is the transcriptome, as well as, the Spi_ERK sequence itself. Is the work clearly and accurately presented and does it cite the current literature? Yes Are sufficient details of materials, methods and analysis provided to allow replication by others? Partly Are all the source data underlying the results available to ensure full reproducibility? Partly Are the conclusions drawn adequately supported by the results? Partly Competing Interests No competing interests were disclosed. Reviewer Expertise Molecular Ecology I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. reply Respond to this report Responses (1) Author Response 03 Jul 2017 Lucile Courtial, Marine Department, Centre Scientifique de Monaco, Monaco, MC-98000, Monaco In this manuscript the authors Courtial et al. validate the use of commercial ERK antibodies for the detection of MAPK-like proteins in Coral. Although the paper is well written and has the quality to be indexed, a few issues should be figure out before its final acceptance. We thank Dr. Parages for her comments and suggestions. To answer her concerns, we have added information and replaced a citation in the manuscript as detailed bellow. In terms of samples preparation and to allow future replication by others researchers would be appropriate to give more information about how to prepare the samples. For example, “Briefly, coral tissue was removed from the skeleton in 1 mL Laemmli buffer” so, how much coral tissue will be dissolved and resupended it in 1 mL of Laemmli Buffer? Less than 0.5 gr? More? All of the tissue from 3-5cm long S. pistillata nubbins was used. We added the precision in the materials and methods: P4: “Two small nubbins (3-5 cm long) were cut off from each mother colony and were allowed to heal for four weeks in 15 L open system tanks before the experiments.” P5: “Briefly, nubbins were airbrushed in 1 mL Laemmli buffer (2% SDS, 10% glycerol, 50mM Tris HCL pH7) ( Laemmli 1970) using an air-pick (5 bars) to remove the totality of the tissues surrounding the skeleton was removed from coral.” While it is true that the authors note that immune-detection were performed as in Courtial et al. (2017), and that DTT (Dithiothreitol) and BFB (Bromophenol blue) were added to the samples and heated (5 minutes at 95°) before loading the gels, it is not specified how tissue extraction was performed. I must assume that the Lysis Buffer used was Laemmli Buffer? And in this case, how they have been unable to detect phosphorylated ERK? As far as I know lysis buffer for phosphorylated proteins usually contents EDTA or EGTA to chelate Mg2+/Ca2+, DTT for reduction of disulfide bonds, serine protease inhibitor (Aprotinin/Leupeptine), phosphatase inhibitors to block dephosphorylation like Na orthovanadate, or Beta-glycerophosphate (false substrate for phosphatase, between others… Coral tissues were indeed lyzed in laemmli buffer. To prevent any doubt when reading the methods, we added a precision in the text: P3: “Briefly, nubbins were airbrushed in 1 mL Laemmli buffer (2% SDS, 10% glycerol, 50mM Tris HCL pH7) (Laemmli 1970) using an air-pick (5 bars) to remove the totality of the tissues surrounding the skeleton.” To answer the concern of Dr. Parages about the possible phosphatase activity in the samples, we would like to specify the fact that, due to a high concentration of SDS, the Laemmli buffer is a strongly denaturing buffer for proteins, including phosphatases. Therefore, this buffer, commonly used at room temperature, preserves phosphorylation of the proteins without the need to add phosphatases inhibitors (see Picco et al. 2016 for example). They also keep everything ice cold? Also, it surprises me that they used 3% low fat milk for membranes blocking, due to also screws up phosphor-tyrosine detection. We fully agree with Dr. Parages, the use of milk as a blocking buffer is usually not recommended for the detection of phospho-proteins as it may contain phospho-proteins that can interact with the anti-phospho primary antibodies. However, we have successfully used this blocking buffer in diverse experimental setups, including human cultured cells, ascidian embryos as well as coral lysates. We used this buffer in the course of this study because it gives far less background noise than any other blocking buffers tested. Regarding the presence of an ERK ortholog in coral, the authors show the sequence with code Spi_isotig05348 (Spi_ERK) has the best hit with the human ERK protein (NP_0011035145) and they make reference to Liew et al., 2014. I was looking for this sequence into this paper, and I could not find it. The authors should provide in which database is the transcriptome, as well as, the Spi_ERK sequence itself. We are grateful to the reviewer for questioning this particular point as it allowed us to uncover a significant error in the citation we used. The reference for the Spi EST containing the ERK orthologue open reading frame was obtained from the database generated during the study by Karako-Lampert, Zoccola et al. (Plos One 2014) and not the one by Liew et al (p.7) . The database containing the sequence can be downloaded from this address: http://data.centrescientifique.mc/CSMdata-stylodata.html . As mentioned in the manuscript, the ERK1 human protein sequence was blasted (tblastn) against the Karako-Lampert database using the blast tool hosted on a publically accessible local server of the Centre Scientifique de Monaco ( http://data.centrescientifique.mc/blast/blast.php ). The correct reference has been added to the manuscript. Due to current major security concerns for our servers, we have chosen not to include the aforementioned URL in the present manuscript. However, this URL is openly disclosed in the Karako-Lampert et al. paper, which should allow readers to access the database without the needing to contact corresponding authors. View more View less Competing Interests No competing interests were disclosed. reply Respond Report a concern Parages ML. Peer Review Report For: Validation of commercial ERK antibodies against the ERK orthologue of the scleractinian coral Stylophora pistillata [version 2; peer review: 3 approved] . F1000Research 2017, 6 :577 ( https://doi.org/10.5256/f1000research.12269.r23149) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/6-577/v1#referee-response-23149 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2017 Castellano I. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 16 Jun 2017 | for Version 1 Immacolata Castellano , Department of Biology and Evolution of Marine Organisms, Stazione Zoologica Anton Dohrn, Naples, Italy 0 Views copyright © 2017 Castellano I. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (1) Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions The paper by Courtial et al. describes the cross-reactivity of two commercial antibodies produced against the mammalian forms of ERK for the scleractinian coral Stylophora pistillata. This should open new perspective for the study of ERK signalling in response to different environmental cues. The paper is clear and well written, however it lacks of some references. In the Introduction, the authors should cite other invertebrates where ERK signalling is known to be conserved and regulated by environmental cues, for example Ciona intestinalis (Castellano et al, PlOS One 2014, Castellano et al, Open Biology 2015). Similarly, in Results and Discussion, when the authors say that .. "a single orthologue of ERK is present in these cnidarians, as opposed to the two genes encoding ERKs in most mammalian genomes”, they should specify that also in other invertebrates, only one ERK form was found (Russo et al, JBC 2004; Castellano et al, PloS One 2014). Also the use of the MEK inhibitor U0126 was assessed in C. intestinalis (Castellano et al, Open Biology 2015). Finally the authors pay attention along the text to some errors, i.e. change “Thr2020/204” with Thr202/204, and “through this medium” with “through this method”. Is the work clearly and accurately presented and does it cite the current literature? Partly Are sufficient details of materials, methods and analysis provided to allow replication by others? Yes Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes Competing Interests No competing interests were disclosed. Reviewer Expertise Biochemistry I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. reply Respond to this report Responses (1) Author Response 03 Jul 2017 Lucile Courtial, Marine Department, Centre Scientifique de Monaco, Monaco, MC-98000, Monaco The paper by Courtial et al. describes the cross-reactivity of two commercial antibodies produced against the mammalian forms of ERK for the scleractinian coral Stylophora pistillata. This should open new perspective for the study of ERK signalling in response to different environmental cues. The paper is clear and well written, however it lacks of some references. We thank Dr. Castellano for her comments and suggestions. We have added information to the manuscript to answer her concerns as detailed bellow. In the Introduction, the authors should cite other invertebrates where ERK signalling is known to be conserved and regulated by environmental cues, for example Ciona intestinalis (Castellano et al, PlOS One 2014, Castellano et al, Open Biology 2015). We added a precision and the reference in the text: “The ERK gene family is evolutionnarily conserved and is found in all eukaryotes, including yeasts, plants, vertebrates and invertebrates (Widmann et al. 1999; Chen et al. 2001; Castellano et al. 2014).” Similarly, in Results and Discussion, when the authors say that .. "a single orthologue of ERK is present in these cnidarians, as opposed to the two genes encoding ERKs in most mammalian genomes”, they should specify that also in other invertebrates, only one ERK form was found (Russo et al, JBC 2004; Castellano et al, PloS One 2014). We added the precision in the text:P4 “This result suggests that a single ortholog of ERK is present in these cnidarians, consistently with previous work where only one ERK ortholog was found (Russo et al. 2004; Castellano et al. 2014) but as opposed to the two genes encoding ERKs in most mammalian genomes (Ip and Davis 1998).” Also the use of the MEK inhibitor U0126 was assessed in C. intestinalis (Castellano et al, Open Biology 2015). Despite extensive search into the reference cited by Dr. Castellano, we could not find any experiment using U0126 in this paper. The aforementioned reference instead reports the use of a dual specificity phosphatase inhibitor. Moreover, the work of Picco et al. (2007) cited in the manuscript already reports the use of U0126 in Ciona embryos. We therefore did not include the suggested citation in the text. Finally the authors pay attention along the text to some errors, i.e. change “Thr2020/204” with Thr202/204, and “through this medium” with “through this method”. We changed the errors in the revised manuscript. View more View less Competing Interests No competing interests were disclosed. reply Respond Report a concern Castellano I. Peer Review Report For: Validation of commercial ERK antibodies against the ERK orthologue of the scleractinian coral Stylophora pistillata [version 2; peer review: 3 approved] . F1000Research 2017, 6 :577 ( https://doi.org/10.5256/f1000research.12269.r23532) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/6-577/v1#referee-response-23532 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2017 Pitzschke A. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 30 May 2017 | for Version 1 Andrea Pitzschke , Department of Cell Biology and Physiology, University of Salzburg, Salzburg, Austria 0 Views copyright © 2017 Pitzschke A. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (1) Approved With Reservations info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions The manuscript provides first insight into a putative MAPK in the coral Stylophora pistillata. Experiments include protein extraction and immunoblot analysis. Overall: The experiments that had been performed are properly designed. However, the manuscript lacks sufficiently-detailed information, as well as controls (protein loading). Conclusions are premature or should be re-phrased. Detailed points of criticism: Title “orthologue” is inappropriate. Should be “homologue”. Methods P3 “small rubbins selected” – please be more specific about size and sampling: “tissue removed from coral”: be more specific. Tissue primarily from the surface, how deep was the cut into the material? (I suggest to include a schematic figure incl. scale-bar). This information is important because inhibitors (e.g. UO126) will only diffuse over a short distance, i.e. not reach deeper layers. P4: “extensive washing”: duration and number of solution changes missing Fig.2B: “% or control” rather OF control. The error bar in the control sample is irrelevant, as it is defined as strictly 100%. There is no documentation of protein loading (e.g. Coomassie-stained membrane after immunodetection). The U126-independent intensity of the ERK-Signal is insufficient as control. Conclusions: “…antibody can be used…in different experimental or environmental conditions” This conclusion is premature. As a minimum, the authors should perform an induction experiment. The inhibitory approach (U126) only evidences that a MAPKK is the upstream regulator. Coral research community will only benefit from the antibody and the current study if dynamic ERK activity responses can be monitored. Is the work clearly and accurately presented and does it cite the current literature? Partly Are sufficient details of materials, methods and analysis provided to allow replication by others? Partly Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Partly Competing Interests No competing interests were disclosed. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. reply Respond to this report Responses (1) Author Response 03 Jul 2017 Lucile Courtial, Marine Department, Centre Scientifique de Monaco, Monaco, MC-98000, Monaco Overall: The experiments that had been performed are properly designed. However, the manuscript lacks sufficiently-detailed information, as well as controls (protein loading). Conclusions are premature or should be re-phrased. We thank Dr. Pitzschke for her comments and suggestions. We have added information to the manuscript and performed an additional experiment to answer her concerns as detailed bellow. Detailed points of criticism: Title “orthologue” is inappropriate. Should be “homologue”. We have used the term “orthologue” in our manuscript in its definition notably given by Walter Fitch (Fitch 1970, 2000), that is: “Orthology is that relationship where sequence divergence follows speciation, that is, where the common ancestor of the two genes lies in the common ancestor of the taxa from which the two sequences were obtained”. Dr. Pitzschke is right, the Spi- and Hs-ERK proteins are homologues but, on top of that, they also are orthologues. We therefore still think that the term “orthologue” is more accurate in our case. Methods P3 “small nubbins selected” –please be more specific about size and sampling: “tissue removed from coral”: be more specific. Tissue primarily from the surface, how deep was the cut into the material? To be more specific, we added the following sentences in the revised manuscript: P2: “Two small nubbins (3-5 cm long) were cut off from each mother colony and were allowed to heal for four weeks in 15 L open system tanks before the experiments.” P3: “Briefly, nubbins were airbrushed in 1 mL Laemmli buffer ( i.e. lysing buffer, 1.5 X, Laemmli 1970) using an air-pick (5 bars) to remove the totality of the tissues surrounding the skeleton was removed from coral.” This information is important because inhibitors (e.g. UO126) will only diffuse over a short distance, i.e. not reach deeper layers. We added a sentence to prevent further doubts concerning the bioavailability of U0126. P6: “In order to test the specificity of the antibodies, we therefore used U0126, a very potent and selective inhibitor of MEK (Bain et al. 2007). The limited thickness of the animal tissue covering the skeleton and the very large surface of contact of both ectoderm and endoderm with the seawater render S. pistillata suitable for treatment with drugs directly diluted in the seawater as we previously showed (Courtial et al. 2017) …” P4: “extensive washing”: duration and number of solution changes missing We added precisions in the materials and methods: P4 : “4 x 30 minutes” Fig.2B: “% or control” rather OF control. The error bar in the control sample is irrelevant, as it is defined as strictly 100%. There is no documentation of protein loading (e.g. Coomassie-stained membrane after immunodetection). The U126-independent intensity of the ERK-Signal is insufficient as control. We added the amido black colored membranes in Figure 2 and Figure S1 as a loading control. Conclusions: “…antibody can be used…in different experimental or environmental conditions” This conclusion is premature. As a minimum, the authors should perform an induction experiment. The inhibitory approach (U126) only evidences that a MAPKK is the upstream regulator. Coral research community will only benefit from the antibody and the current study if dynamic ERK activity responses can be monitored. We performed an additional experiment and added a figure and related text in the manuscript to justify our statement (P9). View more View less Competing Interests No competing interests were disclosed. reply Respond Report a concern Pitzschke A. Peer Review Report For: Validation of commercial ERK antibodies against the ERK orthologue of the scleractinian coral Stylophora pistillata [version 2; peer review: 3 approved] . F1000Research 2017, 6 :577 ( https://doi.org/10.5256/f1000research.12269.r22707) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/6-577/v1#referee-response-22707 Alongside their report, reviewers assign a status to the article: Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions Click here to access the data. The problem Spreadsheet data files may not format correctly if your computer is using different default delimiters (symbols used to separate values into separate cells) - a spreadsheet created in one region is sometimes misinterpreted by computers in other regions. You can change the regional settings on your computer so that the spreadsheet can be interpreted correctly. How to fix it Save downloaded CSV file Open spreadsheet program (e.g. Excel) Click the ‘Data’ tab at the top Click the ‘From text’ icon (top left) Browse for downloaded CSV file, click ‘Import’ Ensure ‘Delimited’ radio button is selected, click ‘Next’ Check one of the appropriate delimiter checkboxes (you can visualize the formatting by looking at the data preview below these options) Click ‘Finish’ Downloaded data do not display as expected? Download the data Dataset citation: Courtial L, Picco V, Pagès G and Ferrier-Pagès C. Dataset 1 in: Validation of commercial ERK antibodies against the ERK orthologue of the scleractinian coral Stylophora pistillata . F1000Research 2017, 6 :577 (https://doi.org/10.5256/f1000research.11365.d159188) Close Click here to access the data. The problem Spreadsheet data files may not format correctly if your computer is using different default delimiters (symbols used to separate values into separate cells) - a spreadsheet created in one region is sometimes misinterpreted by computers in other regions. You can change the regional settings on your computer so that the spreadsheet can be interpreted correctly. How to fix it Save downloaded CSV file Open spreadsheet program (e.g. Excel) Click the ‘Data’ tab at the top Click the ‘From text’ icon (top left) Browse for downloaded CSV file, click ‘Import’ Ensure ‘Delimited’ radio button is selected, click ‘Next’ Check one of the appropriate delimiter checkboxes (you can visualize the formatting by looking at the data preview below these options) Click ‘Finish’ Downloaded data do not display as expected? Download the data Dataset citation: Courtial L, Picco V, Pagès G and Ferrier-Pagès C. Dataset 2 in: Validation of commercial ERK antibodies against the ERK orthologue of the scleractinian coral Stylophora pistillata . F1000Research 2017, 6 :577 (https://doi.org/10.5256/f1000research.11365.d166821) Close Click here to access the data. The problem Spreadsheet data files may not format correctly if your computer is using different default delimiters (symbols used to separate values into separate cells) - a spreadsheet created in one region is sometimes misinterpreted by computers in other regions. You can change the regional settings on your computer so that the spreadsheet can be interpreted correctly. How to fix it Save downloaded CSV file Open spreadsheet program (e.g. Excel) Click the ‘Data’ tab at the top Click the ‘From text’ icon (top left) Browse for downloaded CSV file, click ‘Import’ Ensure ‘Delimited’ radio button is selected, click ‘Next’ Check one of the appropriate delimiter checkboxes (you can visualize the formatting by looking at the data preview below these options) Click ‘Finish’ Downloaded data do not display as expected? Download the data Dataset citation: Courtial L, Picco V, Pagès G and Ferrier-Pagès C. Dataset 3 in: Validation of commercial ERK antibodies against the ERK orthologue of the scleractinian coral Stylophora pistillata . F1000Research 2017, 6 :577 (https://doi.org/10.5256/f1000research.11365.d166825) Close Adjust parameters to alter display View on desktop for interactive features Includes Interactive Elements View on desktop for interactive features Competing Interests Policy Provide sufficient details of any financial or non-financial competing interests to enable users to assess whether your comments might lead a reasonable person to question your impartiality. Consider the following examples, but note that this is not an exhaustive list: Examples of 'Non-Financial Competing Interests' Within the past 4 years, you have held joint grants, published or collaborated with any of the authors of the selected paper. You have a close personal relationship (e.g. parent, spouse, sibling, or domestic partner) with any of the authors. You are a close professional associate of any of the authors (e.g. scientific mentor, recent student). You work at the same institute as any of the authors. You hope/expect to benefit (e.g. favour or employment) as a result of your submission. You are an Editor for the journal in which the article is published. Examples of 'Financial Competing Interests' You expect to receive, or in the past 4 years have received, any of the following from any commercial organisation that may gain financially from your submission: a salary, fees, funding, reimbursements. You expect to receive, or in the past 4 years have received, shared grant support or other funding with any of the authors. You hold, or are currently applying for, any patents or significant stocks/shares relating to the subject matter of the paper you are commenting on. Stay Updated Sign up for content alerts and receive a weekly or monthly email with all newly published articles Register with F1000Research Already registered? Sign in Not now, thanks close PLEASE NOTE If you are an AUTHOR of this article, please check that you signed in with the account associated with this article otherwise we cannot automatically identify your role as an author and your comment will be labelled as a “User Comment”. If you are a REVIEWER of this article, please check that you have signed in with the account associated with this article and then go to your account to submit your report, please do not post your review here. If you do not have access to your original account, please contact us . All commenters must hold a formal affiliation as per our Policies . The information that you give us will be displayed next to your comment. User comments must be in English, comprehensible and relevant to the article under discussion. We reserve the right to remove any comments that we consider to be inappropriate, offensive or otherwise in breach of the User Comment Terms and Conditions . Commenters must not use a comment for personal attacks. When criticisms of the article are based on unpublished data, the data should be made available. I accept the User Comment Terms and Conditions Please confirm that you accept the User Comment Terms and Conditions. Affiliation ✕ refresh Please enter your institution. Note: To add your institution or organisation, start typing the name and then select the correct name from the list. Where applicable, the name will appear in both the original language and in English. Do not paste in the name. If the name does not appear in the drop-down list, we will display the information you have entered. ✕ refresh Country/Region * USA UK Canada China France Germany Afghanistan Aland Islands Albania Algeria American Samoa Andorra Angola Anguilla Antarctica Antigua and Barbuda Argentina Armenia Aruba Australia Austria Azerbaijan Bahamas Bahrain Bangladesh Barbados Belarus Belgium Belize Benin Bermuda Bhutan Bolivia Bosnia and Herzegovina Botswana Bouvet Island Brazil British Indian Ocean Territory British Virgin Islands Brunei Bulgaria Burkina Faso Burundi Cambodia Cameroon Canada Cape Verde Cayman Islands Central African Republic Chad Chile China Christmas Island Cocos (Keeling) Islands Colombia Comoros Congo Cook Islands Costa Rica Cote d'Ivoire Croatia Cuba Cyprus Czech Republic Democratic Republic of the Congo Denmark Djibouti Dominica Dominican Republic Ecuador Egypt El Salvador Equatorial Guinea Eritrea Estonia Ethiopia Falkland Islands Faroe Islands Federated States of Micronesia Fiji Finland France French Guiana French Polynesia French Southern Territories Gabon Georgia Germany Ghana Gibraltar Greece Greenland Grenada Guadeloupe Guam Guatemala Guernsey Guinea Guinea-Bissau Guyana Haiti Heard Island and Mcdonald Islands Holy See (Vatican City State) Honduras Hong Kong Hungary Iceland India Indonesia Iran Iraq Ireland Israel Italy Jamaica Japan Jersey Jordan Kazakhstan Kenya Kiribati Kosovo (Serbia and Montenegro) Kuwait Kyrgyzstan Lao People's Democratic Republic Latvia Lebanon Lesotho Liberia Libya Liechtenstein Lithuania Luxembourg Macao Madagascar Malawi Malaysia Maldives Mali Malta Marshall Islands Martinique Mauritania Mauritius Mayotte Mexico Minor Outlying Islands of the United States Moldova Monaco Mongolia Montenegro Montserrat Morocco Mozambique Myanmar Namibia Nauru Nepal Netherlands Antilles New Caledonia New Zealand Nicaragua Niger Nigeria Niue Norfolk Island North Korea North Macedonia Northern Mariana Islands Norway Oman Pakistan Palau Palestinian Territory Panama Papua New Guinea Paraguay Peru Philippines Pitcairn Poland Portugal Puerto Rico Qatar Reunion Romania Russian Federation Rwanda Saint Helena Saint Kitts and Nevis Saint Lucia Saint Pierre and Miquelon Saint Vincent and the Grenadines Samoa San Marino Sao Tome and Principe Saudi Arabia Senegal Serbia Seychelles Sierra Leone Singapore Slovakia Slovenia Solomon Islands Somalia South Africa South Georgia and the South Sandwich Is South Korea South Sudan Spain Sri Lanka Sudan Suriname Svalbard and Jan Mayen Swaziland Sweden Switzerland Syria Taiwan Tajikistan Tanzania Thailand The Gambia The Netherlands Timor-Leste Togo Tokelau Tonga Trinidad and Tobago Tunisia Turkey Turkmenistan Turks and Caicos Islands Tuvalu UK USA Uganda Ukraine United Arab Emirates United States Virgin Islands Uruguay Uzbekistan Vanuatu Venezuela Vietnam Wallis and Futuna West Bank and Gaza Strip Western Sahara Yemen Zambia Zimbabwe Please select your country/region. You must enter a comment. Competing Interests Please disclose any competing interests that might be construed to influence your judgment of the article's or peer review report's validity or importance. Competing Interests Policy Provide sufficient details of any financial or non-financial competing interests to enable users to assess whether your comments might lead a reasonable person to question your impartiality. Consider the following examples, but note that this is not an exhaustive list: Examples of 'Non-Financial Competing Interests' Within the past 4 years, you have held joint grants, published or collaborated with any of the authors of the selected paper. You have a close personal relationship (e.g. parent, spouse, sibling, or domestic partner) with any of the authors. You are a close professional associate of any of the authors (e.g. scientific mentor, recent student). You work at the same institute as any of the authors. You hope/expect to benefit (e.g. favour or employment) as a result of your submission. You are an Editor for the journal in which the article is published. Examples of 'Financial Competing Interests' You expect to receive, or in the past 4 years have received, any of the following from any commercial organisation that may gain financially from your submission: a salary, fees, funding, reimbursements. You expect to receive, or in the past 4 years have received, shared grant support or other funding with any of the authors. You hold, or are currently applying for, any patents or significant stocks/shares relating to the subject matter of the paper you are commenting on. Please state your competing interests The comment has been saved. An error has occurred. Please try again. Cancel Post var lTitle = "Validation of commercial ERK antibodies against...".replace("'", ''); var linkedInUrl = "http://www.linkedin.com/shareArticle?url=https://f1000research.com/articles/6-577/v2" + "&title=" + encodeURIComponent(lTitle) + "&summary=" + encodeURIComponent('Read the article by '); var deliciousUrl = "https://del.icio.us/post?url=https://f1000research.com/articles/6-577/v2&title=" + encodeURIComponent(lTitle); var redditUrl = "http://reddit.com/submit?url=https://f1000research.com/articles/6-577/v2" + "&title=" + encodeURIComponent(lTitle); linkedInUrl += encodeURIComponent('Courtial L et al.'); var offsetTop = /chrome/i.test( navigator.userAgent ) ? 4 : -10; var addthis_config = { ui_offset_top: offsetTop, services_compact : "facebook,twitter,www.linkedin.com,www.mendeley.com,reddit.com", services_expanded : "facebook,twitter,www.linkedin.com,www.mendeley.com,reddit.com", services_custom : [ { name: "LinkedIn", url: linkedInUrl, icon:"/img/icon/at_linkedin.svg" }, { name: "Mendeley", url: "http://www.mendeley.com/import/?url=https://f1000research.com/articles/6-577/v2/mendeley", icon:"/img/icon/at_mendeley.svg" }, { name: "Reddit", url: redditUrl, icon:"/img/icon/at_reddit.svg" }, ] }; var addthis_share = { url: "https://f1000research.com/articles/6-577", templates : { twitter : "Validation of commercial ERK antibodies against the ERK orthologue.... Courtial L et al., published by " + "@F1000Research" + ", https://f1000research.com/articles/6-577/v2" } }; if (typeof(addthis) != "undefined"){ addthis.addEventListener('addthis.ready', checkCount); addthis.addEventListener('addthis.menu.share', checkCount); } $(".f1r-shares-twitter").attr("href", "https://twitter.com/intent/tweet?text=" + addthis_share.templates.twitter); $(".f1r-shares-facebook").attr("href", "https://www.facebook.com/sharer/sharer.php?u=" + addthis_share.url); $(".f1r-shares-linkedin").attr("href", addthis_config.services_custom[0].url); $(".f1r-shares-reddit").attr("href", addthis_config.services_custom[2].url); $(".f1r-shares-mendelay").attr("href", addthis_config.services_custom[1].url); function checkCount(){ setTimeout(function(){ $(".addthis_button_expanded").each(function(){ var count = $(this).text(); if (count !== "" && count != "0") $(this).removeClass("is-hidden"); else $(this).addClass("is-hidden"); }); }, 1000); } close How to cite this report {{reportCitation}} Cancel Copy Citation Details $(function(){R.ui.buttonDropdowns('.dropdown-for-downloads');}); $(function(){R.ui.toolbarDropdowns('.toolbar-dropdown-for-downloads');}); $.get("/articles/acj/11365/13040") new F1000.Clipboard(); new F1000.ThesaurusTermsDisplay("articles", "article", "13040"); $(document).ready(function() { $( "#frame1" ).on('load', function() { var mydiv = $(this).contents().find("div"); var h = mydiv.height(); console.log(h) }); var tooltipLivingFigure = jQuery(".interactive-living-figure-label .icon-more-info"), titleLivingFigure = tooltipLivingFigure.attr("title"); tooltipLivingFigure.simpletip({ fixed: true, position: ["-115", "30"], baseClass: 'small-tooltip', content:titleLivingFigure + " " }); tooltipLivingFigure.removeAttr("title"); $("body").on("click", ".cite-living-figure", function(e) { e.preventDefault(); var ref = $(this).attr("data-ref"); $(this).closest(".living-figure-list-container").find("#" + ref).fadeIn(200); }); $("body").on("click", ".close-cite-living-figure", function(e) { e.preventDefault(); $(this).closest(".popup-window-wrapper").fadeOut(200); }); $(document).on("mouseup", function(e) { var metricsContainer = $(".article-metrics-popover-wrapper"); if (!metricsContainer.is(e.target) && metricsContainer.has(e.target).length === 0) { $(".article-metrics-close-button").click(); } }); var articleId = $('#articleId').val(); if($("#main-article-count-box").attachArticleMetrics) { $("#main-article-count-box").attachArticleMetrics(articleId, { articleMetricsView: true }); } }); var figshareWidget = $(".new_figshare_widget"); if (figshareWidget.length > 0) { window.figshare.load("f1000", function(Widget) { // Select a tag/tags defined in your page. In this tag we will place the widget. _.map(figshareWidget, function(el){ var widget = new Widget({ articleId: $(el).attr("figshare_articleId") //height:300 // this is the height of the viewer part. [Default: 550] }); widget.initialize(); // initialize the widget widget.mount(el); // mount it in a tag that's on your page // this will save the widget on the global scope for later use from // your JS scripts. This line is optional. //window.widget = widget; }); }); } close Error Close Add Reset F1000.MICROSERVICES.AFFILIATION = ''; $(document).ready(function () { $('.js-affiliations-form').each((index, form) => { new AffiliationForm({ formId: form.id, institutionErrorSelector: '.comment-enter-institution', departmentErrorSelector: '.comment-enter-department', placeSelector: '.js-add-comment-place', stateSelector: '.js-add-comment-state', zipCodeSelector: '.js-add-comment-zipcode', countrySelector: '.js-add-comment-country', countryErrorSelector: '.comment-enter-country', }); }); }); $(document).ready(function () { var reportIds = { "23147": 0, "22955": 0, "23148": 0, "22956": 0, "23532": 11, "23149": 19, "23150": 0, "23151": 0, "22258": 0, "22610": 0, "22259": 0, "22611": 0, "22707": 16, "22260": 0, "22612": 0, "23988": 0, "24020": 9, "22261": 0, "23989": 0, "22262": 0, "23990": 9, "22263": 0, }; $(".referee-response-container,.js-referee-report").each(function(index, el) { var reportId = $(el).attr("data-reportid"), reportCount = reportIds[reportId] || 0; $(el).find(".comments-count-container,.js-referee-report-views").html(reportCount); }); var uuidInput = $("#article_uuid"), oldUUId = uuidInput.val(), newUUId = "680b1914-700a-4b6b-acd0-de029f8407d3"; uuidInput.val(newUUId); $("a[href*='article_uuid=']").each(function(index, el) { var newHref = $(el).attr("href").replace(oldUUId, newUUId); $(el).attr("href", newHref); }); }); An innovative open access publishing platform offering rapid publication and open peer review, whilst supporting data deposition and sharing. Browse Gateways Collections How it Works Contact For Developers Cookie Notice Privacy Notice RSS Submit Your Research Follow us © 2012-2026 F1000 Research Ltd. ISSN 2046-1402 | Legal | Partner of Research4Life • CrossRef • ORCID • FAIRSharing R.templateTests.simpleTemplate = R.template(' $text $text $text $text $text '); R.templateTests.runTests(); var F1000platform = new F1000.Platform({ name: "f1000research", displayName: "F1000Research", hostName: "f1000research.com", id: "1", editorialEmail: "[email protected]", infoEmail: "[email protected]", usePmcStats: true }); $(function(){R.ui.dropdowns('.dropdown-for-authors, .dropdown-for-about, .dropdown-for-myresearch');}); // $(function(){R.ui.dropdowns('.dropdown-for-referees');}); $(document).ready(function () { if ($(".cookie-warning").is(":visible")) { $(".sticky").css("margin-bottom", "35px"); $(".devices").addClass("devices-and-cookie-warning"); } $(".cookie-warning .close-button").click(function (e) { $(".devices").removeClass("devices-and-cookie-warning"); $(".sticky").css("margin-bottom", "0"); }); $("#tweeter-feed .tweet-message").each(function (i, message) { var self = $(message); self.html(linkify(self.html())); }); $(".partner").on("mouseenter mouseleave", function() { $(this).find(".gray-scale, .colour").toggleClass("is-hidden"); }); }); Sign In Remember me Forgotten your password? Sign In Cancel Email or password not correct. Please try again Please wait... $(function(){ // Note: All the setup needs to run against a name attribute and *not* the id due the clonish // nature of facebox... $("a[id=googleSignInButton]").click(function(event){ event.preventDefault(); $("input[id=oAuthSystem]").val("GOOGLE"); $("form[id=oAuthForm]").submit(); }); $("a[id=facebookSignInButton]").click(function(event){ event.preventDefault(); $("input[id=oAuthSystem]").val("FACEBOOK"); $("form[id=oAuthForm]").submit(); }); $("a[id=orcidSignInButton]").click(function(event){ event.preventDefault(); $("input[id=oAuthSystem]").val("ORCID"); $("form[id=oAuthForm]").submit(); }); }); If you've forgotten your password, please enter your email address below and we'll send you instructions on how to reset your password. The email address should be the one you originally registered with F1000. Email address not valid, please try again You registered with F1000 via Google, so we cannot reset your password. To sign in, please click here . If you still need help with your Google account password, please click here . You registered with F1000 via Facebook, so we cannot reset your password. To sign in, please click here . If you still need help with your Facebook account password, please click here . Code not correct, please try again Reset password Cancel Email us for further assistance. Server error, please try again. If your email address is registered with us, we will email you instructions to reset your password. If you think you should have received this email but it has not arrived, please check your spam filters and/or contact for further assistance. Please wait... Register $(document).ready(function () { signIn.createSignInAsRow($("#sign-in-form-gfb-popup")); $(".target-field").each(function () { var uris = $(this).val().split("/"); if (uris.pop() === "login") { $(this).val(uris.toString().replace(",","/")); } }); });