Identification of Orb2-interacting proteins from the adult Drosophila brain.

(A) Schematic of the experimental design. (B) Representative examples of silver-stained gels of anti-HA IP of Orb2BHA (left panel) or Orb2AHA (middle panel) and anti-Orb2 IP of Orb2AHA (right panel) used for proteomic analysis. The overexpressed monomeric Orb2A and Orb2B proteins are visible in the silver-stained gels. The wild-type flies serve as a control for HA transgene, and purified guinea pig IgG serves as a control for anti-Orb2 antibody. (C) Anti-Orb2 antibody immunopurified both monomeric and amyloid-like oligomeric Orb2 from Orb2AHA expressing fly head extracts. (D) The distribution of 61 proteins that were significantly enriched in the Orb2 immunoprecipitates over control. (E) Representative IP–Western blots of candidate proteins that were tested for pair-wise interaction with Orb2 in S2 cells. The FLAG-tagged putative candidate proteins were coexpressed with untagged Orb2 proteins, immunoprecipitated with anti-FLAG antibodies, and Western blotted with anti-Orb2 antibody. The proteins indicated in green have consistently shown above background binding. The flotilin (Flo2) gene, indicated in red, was overrepresented but not significantly enriched in Orb2 immunoprecipitate. However, it binds to Orb2A. The asterisk indicates the position of candidate proteins in SDS-PAGE. The arrows indicate anti-FLAG antibody cross-reacting polypeptides in S2 cell extracts. Unless indicated otherwise, in IP experiments 5% of the lysate is used as loading controls. (F). Gene Ontology (GO) enrichment analysis of Orb2 proteome. The FBgn IDs of the candidate Orb2-interactors were submitted to DAVID web server. Selected nonredundant terms are shown. The p values and fold enrichments were determined using the Drosophila melanogaster genome as background. Uncorrected p values are shown due to prior filtering for enriched peptides and the small input sample size. Also see Figure S1 and Table S1.