ANKIB1 Functions as a Partner of E3 ubiquitin ligase 

ANKIB1 Functions as a Partner of E3 ubiquitin ligase  | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (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;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article ANKIB1 Functions as a Partner of E3 ubiquitin ligase Bohong Xie, Zhenling Ma, Zishan Yang, Yuanbo Li, Shirao Liu, Haijie Yang, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-232896/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract ANKIB1 is an RBR domain-containing protein. It has been classified as an E3 ubiquitin ligase for a long time. However, little is known about its E3 ligase activity and related ubiquitin-conjugating enzymes (E2s). We investigated the expression pattern, cellular localization, and brain distribution of ANKIB1. Immunoblotting showed that human cancer cell lines had different expression patterns of ANKIB1 with virus-transformed immortal lymphocytes and human brain. Immunocytofluorescence analysis indicated that ANKIB1 was mainly localized in the cytoplasm, and immunohistochemistry staining demonstrated that ANKIB1 was highly expressed in the rat brain cortex but seldom expressed in the basal ganglia. We then performed coimmunoprecipitation, in vitro and in vivo ubiquitination assay to determine whether ANKIB1 functions as an E3 ligase. A panel of class I and class III E2s were tested, of which UbcH8, UbcH10, and UbcH13 were confirmed to interact with ANKIB1. Moreover, ANKIB1 showed E3 ligase activity both in vivo and in vitro, but it promoted protein ubiquitination more efficiently in vivo. These data suggest that ANKIB1 acts as a partner of an E3 ligase complex, and may play important roles in regulating tumorigenesis and brain cortex function. Molecular Biology ANKIB1 E3 ligase RBR ubiquitination ubiquitin-conjugating enzyme Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Full Text Due to technical limitations, full-text HTML conversion of this manuscript could not be completed. However, the latest manuscript can be downloaded and accessed as a PDF. Tables Table 1 Primers for PCR Name Sequence (5′→3′) ANK-NBF ANK-NBR RBR-F RBR-R Ub-FL Ub-FS Ub-RL Ub-RS H5B-F H5B-R H6-FL H6-FS H6-RL H6-RS H7-F H7-R H8-F H8-R ATTCCGTAAAGCACTCAT AACTGGTGTCTAAATCCC AGGATCCACCAGTTTGTGTGAC TCTCGAGTCAGACTTCATAGCGAGTAC TATGCAGATCTTCGTGAAGACCC TGCAGATCTTCGTGAAGACCC TCGAGACCACCTCTCAGACGCAGGAC GACCACCTCTCAGACGCAGGAC CTCATCTGAAGCTTGCTCTGAAGAGAATC ATCCTTACATCGCATACTTCTGAGTCCAT AATTCGGATGACGATTCGAGGGC CGGATGACGATTCGAGGGC TCGACTTATGTAGCGTATCTCTTGGTC CTTATGTAGCGTATCTCTTGGTC ATCTTGGAAAGCTTGCGGCCAGCAGGAG ATCGCGGCCGCTCGAGTTAGTC ATATGGCCAAGCTTATGGCGAGCATGCGAG ATCTTTAGGAGGGCCGGTCCACT Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-232896","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":12569404,"identity":"0b7a977a-b9ec-45d1-ae14-0724edc16198","order_by":0,"name":"Bohong Xie","email":"","orcid":"","institution":"Xinxiang Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Bohong","middleName":"","lastName":"Xie","suffix":""},{"id":12569405,"identity":"d816491e-cb4a-4167-a1e5-f9a8272c4de8","order_by":1,"name":"Zhenling Ma","email":"","orcid":"","institution":"Henan Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhenling","middleName":"","lastName":"Ma","suffix":""},{"id":12569406,"identity":"b325f200-10ab-4ca9-b16d-861ab32bab75","order_by":2,"name":"Zishan Yang","email":"","orcid":"","institution":"Xinxiang Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zishan","middleName":"","lastName":"Yang","suffix":""},{"id":12569407,"identity":"d67be00e-0ec9-4d48-b8c4-3980e461dc50","order_by":3,"name":"Yuanbo Li","email":"","orcid":"","institution":"Xinxiang Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yuanbo","middleName":"","lastName":"Li","suffix":""},{"id":12569408,"identity":"89dc4025-755d-452b-9204-f95f26ce0687","order_by":4,"name":"Shirao Liu","email":"","orcid":"","institution":"Xinxiang Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Shirao","middleName":"","lastName":"Liu","suffix":""},{"id":12569409,"identity":"3ef77b9b-73e0-4b1f-867c-eeafb8a5e90f","order_by":5,"name":"Haijie Yang","email":"","orcid":"","institution":"Xinxiang Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Haijie","middleName":"","lastName":"Yang","suffix":""},{"id":12569410,"identity":"4ba16ddf-270f-4f5e-8e65-01fb028d8218","order_by":6,"name":"Lei Wang","email":"","orcid":"","institution":"Xinxiang Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Lei","middleName":"","lastName":"Wang","suffix":""},{"id":12569411,"identity":"2fc7b03b-89a5-4ecc-a5e6-c1dd9313dd28","order_by":7,"name":"Binfeng Cheng","email":"","orcid":"","institution":"Xinxiang Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Binfeng","middleName":"","lastName":"Cheng","suffix":""},{"id":12569412,"identity":"0d5c1bc7-ad7e-4239-ad3c-1736c9f9f6b1","order_by":8,"name":"Zhiwei Feng","email":"","orcid":"","institution":"Xinxiang Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhiwei","middleName":"","lastName":"Feng","suffix":""},{"id":12569413,"identity":"37a26181-3d01-4280-9616-58723deceb52","order_by":9,"name":"Yunfei Xie","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1UlEQVRIiWNgGAWjYDACZgjFz8DMfODAhwoStEg2MLMlHpxxhgTLJBsYeIwP87YQodTgOPMBZt62OxL87DwfDvA2MMjzix0gYHozWwJQyzMJyWbeDQckdzAYzpydgF8LPzOPAVDL4TqDw0AthmcYEgxuE9DCxsz/AaRFwv4wz4MDiW1EaAHawgDWYgBkHDhIjBagXwwY55w7LCFxmM3gYMMZCcJ+MTh/+AHDm7LDEvz9hx9//lNhI88vTUALELD/4kFwJAgqBwPGH8SpGwWjYBSMgpEKAMz+P7r4ndqxAAAAAElFTkSuQmCC","orcid":"","institution":"Xinxiang Medical University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Yunfei","middleName":"","lastName":"Xie","suffix":""}],"badges":[],"createdAt":"2021-02-11 06:24:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-232896/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-232896/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":6332141,"identity":"1162047c-fda2-4da9-9764-506d851d5a79","added_by":"auto","created_at":"2021-02-24 22:34:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":10312204,"visible":true,"origin":"","legend":"Structure of ANKIB1 protein. (a) Schematic representation of human ANKIB1 protein. The structure was predicted by SMART/Pfam with manual correction. CC, coiled-coil motif. LZ, leucine zipper motif. (b) Multiple sequence alignment of ANKIB1 orthologs from human (hANKIB1, Q9P2G1), mouse (mANKIB1, NP_001276456), emperor penguin (pANKIB1, XP_009273943), alligator mississippiensis (aANKIB1, XP_006258998), and zebrafish (zANKIB1, NP_001074461). Numbers indicate ANKIB1 amino acid positions. Consensus amino acid residues were arranged in the top lines. Conservative cysteine (#) and histidine (▼) residues are indicated in the RBR supradomain, and key residues of the leucine zipper are also labeled (♦). ","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-232896/v1/c14da153afd2662d63c27d9e.png"},{"id":6331715,"identity":"4e9a4b99-b22f-4cec-bb73-8369e8262859","added_by":"auto","created_at":"2021-02-24 22:31:52","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":86816,"visible":true,"origin":"","legend":"Analysis of the ANKIB1 protein isoforms and mRNA variants. (a) Immunoblotting analysis of ANKIB1 protein isoforms in human immortal cell lines. The whole cell lysate of each cell line containing 50 μg proteins was subjected to immunoblotting with the anti-ANKIB1 mAb as the primary antibody. (b) Northern blotting analysis of ANKIB1 mRNA variants in SH-SY5Y cells. ANKIB1-specific cDNA probe was synthesized by PCR and labeled with digoxigenin. Total RNA was isolated by guanidine isothiocyanate extraction from the SH-SY5Y cells and hybridized with ANKIB1 cDNA probe. Plasmid pCMV-Myc-ANKIB1(1-480) served as a positive control.","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-232896/v1/186fe1152886ca049bcb8671.png"},{"id":6331686,"identity":"c693b877-f197-4d69-8e03-7e5ac042bc8d","added_by":"auto","created_at":"2021-02-24 22:31:50","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":225913,"visible":true,"origin":"","legend":"Localization of ANKIB1 in HEK293T cells. (a) Immunofluorescence staining of endogenous ANKIB1 in HEK293T cells. HEK293T cells were plated on glass coverslips. 24 h later, the cells were fixed and processed for immunofluorescence staining. Endogenous ANKIB1 was detected by using the anti-ANKIB1 mAb (green). The nucleus was visualized in blue using DAPI staining. (b) Distribution of ANKIB1 isoforms in the cytoplasm and nucleus of HEK293T cells. The cytoplasmic and nuclear proteins were extracted and subjected to immunoblotting with the anti-ANKIB1 mAb. The gray-scale ratios of the 122 kDa isoform to the 88 kDa isoform were calculated.","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-232896/v1/94f33f59da996e8fe4fe96ec.png"},{"id":6331681,"identity":"d3451708-953b-4370-beb2-7b007c320316","added_by":"auto","created_at":"2021-02-24 22:31:49","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":728473,"visible":true,"origin":"","legend":"ANKIB1 expression detected by immunohistochemistry in rat brain cortex. (a) and (b) ANKIB1 expression in the brain cortex. (c) ANKIB1 expression in the basal ganglia. (d) Enlarged images of (c). Positive cells were defined as presenting buffy grains in cytoplasmas shown by arrows. Scalar bars present 20 μm in (a), 10 μm in (b) and (d), while 50 μm in (c).\n\n","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-232896/v1/b6e985987c43a8ce351f5e6c.png"},{"id":6331605,"identity":"ededaa89-76e8-4a20-8fd5-94bed577432f","added_by":"auto","created_at":"2021-02-24 22:31:46","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":156146,"visible":true,"origin":"","legend":"Association of ANKIB1 with UbcH8, UbcH10, and UbcH13. (a) Interaction between overexpressed ANKIB1 and E2s. 9 µg pCMV-Myc-ANKIB1 was co-transfected with 6 µg of every expression vector encoding FLAG-Ubc into HEK293T cells. Cells were lysed 48 h after transfection, and the lysates were subjected to immunoprecipitation with an anti-Myc antibody. The precipitates were resolved on SDS-PAGE gel and subjected to immunoblotting with anti-FLAG or anti-Myc antibody. (b) Interaction between endogenous ANKIB1 and overexpressed E2s. HEK293T cells were transfected with 6 µg of pFLAG-CMV-UbcH8, pCMV-FLAG-UbcH10, or pCMV-FLAG-UbcH13. Immunoprecipitates prepared by anti-FLAG antibody were subjected to immunoblotting with anti-FLAG mAb or anti-ANKIB1 polyclonal antibody.","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-232896/v1/b225f912c5028d1175e0222d.png"},{"id":6332161,"identity":"f1e4e98b-4c1b-4386-b682-c6ea35e1935c","added_by":"auto","created_at":"2021-02-24 22:34:52","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":142645,"visible":true,"origin":"","legend":"ANKIB1 is involved in E2-dependent self-ubiquitination both in vitro and in vivo. (a) In vitro ubiquitination assay. His6-ANKIB1RBR was incubated with recombinant His6-Ub, E1, and E2s at 30℃ for 1 h. The products were resolved by SDS-PAGE and immunoblotted with an anti-Ub antibody. (b) In vitro ubiquitination assay. GST-ANKIB1(1-480) was incubated with recombinant His6-Ub, E1, and E2s at 30℃ for 1 h. The products were resolved by SDS-PAGE and immunoblotted with an anti-Ub antibody. (c) In vivo ubiquitination assay. HEK293T cells were co-transfected with pCMV-Myc-ANKIB1 and pRK5-HA-Ub and incubated for 40 h in the presence of proteasome inhibitor, MG132. The lysates were subjected to immunoprecipitation with an anti-Myc antibody and immunoblotted with anti-HA or anti-Myc antibody. ","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-232896/v1/d7b9bb7d44baf07c9d251e76.png"},{"id":13595402,"identity":"58da9971-a601-41ad-8a9a-e60d95420865","added_by":"auto","created_at":"2021-09-17 05:24:19","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":685008,"visible":true,"origin":"","legend":"","description":"","filename":"ANKIB1functionsasapartnerofE3ubiquitinligase.pdf","url":"https://assets-eu.researchsquare.com/files/rs-232896/v1_covered.pdf"},{"id":6332119,"identity":"1ad3d0fb-21df-49d9-8873-e29d8b404982","added_by":"auto","created_at":"2021-02-24 22:34:43","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":306369,"visible":true,"origin":"","legend":"","description":"","filename":"ANKIB1functionsasapartnerofE3ubiquitinligase.pdf","url":"https://assets-eu.researchsquare.com/files/rs-232896/v1_stamped.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eANKIB1 Functions as a Partner of E3 ubiquitin ligase\u0026nbsp;\u003c/p\u003e","fulltext":[{"header":"Full Text","content":"Due to technical limitations, full-text HTML conversion of this manuscript could not be completed. However, the latest manuscript can be downloaded and \u003ca href='/article/rs-232896/latest.pdf' target='_blank'\u003e accessed as a PDF.\u003c/a\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1\u0026nbsp; Primers for PCR\u003c/p\u003e\n\u003ctable border=\"1\" width=\"0\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd width=\"97\"\u003e\n\u003cp\u003eName\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"281\"\u003e\n\u003cp\u003eSequence (5\u0026prime;\u0026rarr;3\u0026prime;)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"97\"\u003e\n\u003cp\u003eANK-NBF\u003c/p\u003e\n\u003cp\u003eANK-NBR\u003c/p\u003e\n\u003cp\u003eRBR-F\u003c/p\u003e\n\u003cp\u003eRBR-R\u003c/p\u003e\n\u003cp\u003eUb-FL\u003c/p\u003e\n\u003cp\u003eUb-FS\u003c/p\u003e\n\u003cp\u003eUb-RL\u003c/p\u003e\n\u003cp\u003eUb-RS\u003c/p\u003e\n\u003cp\u003eH5B-F\u003c/p\u003e\n\u003cp\u003eH5B-R\u003c/p\u003e\n\u003cp\u003eH6-FL\u003c/p\u003e\n\u003cp\u003eH6-FS\u003c/p\u003e\n\u003cp\u003eH6-RL\u003c/p\u003e\n\u003cp\u003eH6-RS\u003c/p\u003e\n\u003cp\u003eH7-F\u003c/p\u003e\n\u003cp\u003eH7-R\u003c/p\u003e\n\u003cp\u003eH8-F\u003c/p\u003e\n\u003cp\u003eH8-R\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"281\"\u003e\n\u003cp\u003eATTCCGTAAAGCACTCAT\u003c/p\u003e\n\u003cp\u003eAACTGGTGTCTAAATCCC\u0026nbsp;\u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAGGATCCACCAGTTTGTGTGAC\u003c/p\u003e\n\u003cp\u003eTCTCGAGTCAGACTTCATAGCGAGTAC\u003c/p\u003e\n\u003cp\u003eTATGCAGATCTTCGTGAAGACCC\u003c/p\u003e\n\u003cp\u003eTGCAGATCTTCGTGAAGACCC\u003c/p\u003e\n\u003cp\u003eTCGAGACCACCTCTCAGACGCAGGAC\u003c/p\u003e\n\u003cp\u003eGACCACCTCTCAGACGCAGGAC\u003c/p\u003e\n\u003cp\u003eCTCATCTGAAGCTTGCTCTGAAGAGAATC\u003c/p\u003e\n\u003cp\u003eATCCTTACATCGCATACTTCTGAGTCCAT\u003c/p\u003e\n\u003cp\u003eAATTCGGATGACGATTCGAGGGC\u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCGGATGACGATTCGAGGGC\u003c/p\u003e\n\u003cp\u003eTCGACTTATGTAGCGTATCTCTTGGTC\u003c/p\u003e\n\u003cp\u003eCTTATGTAGCGTATCTCTTGGTC\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eATCTTGGAAAGCTTGCGGCCAGCAGGAG\u003c/p\u003e\n\u003cp\u003eATCGCGGCCGCTCGAGTTAGTC\u003c/p\u003e\n\u003cp\u003eATATGGCCAAGCTTATGGCGAGCATGCGAG\u003c/p\u003e\n\u003cp\u003eATCTTTAGGAGGGCCGGTCCACT\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"ANKIB1, E3 ligase, RBR, ubiquitination, ubiquitin-conjugating enzyme ","lastPublishedDoi":"10.21203/rs.3.rs-232896/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-232896/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eANKIB1 is an RBR domain-containing protein. It has been classified as an E3 ubiquitin ligase for a long time. However, little is known about its E3 ligase activity and related ubiquitin-conjugating enzymes (E2s). We investigated the expression pattern, cellular localization, and brain distribution of ANKIB1. Immunoblotting showed that human cancer cell lines had different expression patterns of ANKIB1 with virus-transformed immortal lymphocytes and human brain. Immunocytofluorescence analysis indicated that ANKIB1 was mainly localized in the cytoplasm, and immunohistochemistry staining demonstrated that ANKIB1 was highly expressed in the rat brain cortex but seldom expressed in the basal ganglia. We then performed coimmunoprecipitation, in vitro and in vivo ubiquitination assay to determine whether ANKIB1 functions as an E3 ligase. A panel of class I and class III E2s were tested, of which UbcH8, UbcH10, and UbcH13 were confirmed to interact with ANKIB1. Moreover, ANKIB1 showed E3 ligase activity both in vivo and in vitro, but it promoted protein ubiquitination more efficiently in vivo. These data suggest that ANKIB1 acts as a partner of an E3 ligase complex, and may play important roles in regulating tumorigenesis and brain cortex function.\u0026nbsp;\u003c/p\u003e","manuscriptTitle":"ANKIB1 Functions as a Partner of E3 ubiquitin ligase\u0026nbsp;","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-02-24 22:31:13","doi":"10.21203/rs.3.rs-232896/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b7b0eece-471b-4173-9e6c-3bc8ea72ac21","owner":[],"postedDate":"February 24th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":2593335,"name":"Molecular Biology"}],"tags":[],"updatedAt":"2021-02-24T22:31:39+00:00","versionOfRecord":[],"versionCreatedAt":"2021-02-24 22:31:13","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-232896","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-232896","identity":"rs-232896","version":["v1"]},"buildId":"FbvkV6FR0MCFSLy54lSbu","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}