Characterization of Endogenous and Epitope-Tagged Ubiquitin Expression and Ubiquitination Patterns at Drosophila Neuromuscular Junctions

Ubiquitination is a highly regulated post-translational modification that controls protein stability, activity, localization, and interactions. Disruption of this process contributes to a broad range of human diseases, including cancer, neurodegeneration, and immune dysfunction. The genetic model organism Drosophila melanogaster offers powerful tools to study ubiquitination in vivo , where versatile systems such as GAL4/UAS, Split-GAL4, GeneSwitch, and MARCM can be combined with imaging and biochemical approaches to achieve precise spatial and temporal analyses. To fully exploit these advantages, experimental designs often require epitope-tagged UAS-Ubiquitin transgenes—both modified and unmodified—that accurately recapitulate the behavior of endogenous Ubiquitin. Here, we report the generation and validation of a panel of epitope-tagged UAS-Ubiquitin transgenes. We first characterize the endogenous ubiquitination pattern at larval neuromuscular junctions (NMJs), then compare that with transgenically expressed Ubiquitin in muscle. Our analysis showed that transgenic His-Myc-, HA-, and His-Biotin-tagged Ubiquitin proteins accurately reproduce endogenous postsynaptic ubiquitination patterns. These validated transgenes provide versatile resources for investigating ubiquitination dynamics across diverse cellular and developmental contexts.