Fig 1. Strategy for identification of Urm1 conjugation targets in Drosophila embryos, larvae and adults in vivo.

A. Schematic representation of the rationale for identifying Urm1-binding proteins and thereby candidate targets of urmylation during three critical stages of Drosophila development; embryogenesis, late larval stages and adulthood. Shortly, Flag-Urm1 associated proteins were enriched by immunoprecipitation with Flag M2 magnetic beads, which subsequently were subjected to on-bead trypsin digestion, followed by mass spectrometry and identification by standard bioinformatics analysis. B. Western blot illustrating the distribution of candidate Urm1 targets in embryos (left panel), larvae (middle panel) and adults (right panel), respectively. Urm1-interacting proteins were captured in the presence of NEM by Flag M2 immunoprecipitation, using magnetic beads, resolved under denaturing conditions by SDS-PAGE and detected by anti-Urm1 antibodies (* depicts endoge^◆nous Urm1 expressed in control Actin5C>w¹¹¹⁸ samples and indicates the unconjugated Flag-Urm1 fusion protein). Input represents 30 μg of the total lysate of the indicated genotypes.