Proteomic Analysis of BEAF-32B Interactome

A P-element based transgenic fly line with an insulated FLAG-BEAF-32B-EGFP gene expressed from its endogenous promoter (Avva and Hart 2016 (link)) on the X chromosome was used to collect 4- to 20-h embryos. These flies also had the wild-type BEAF gene. Control embryos were collected from y¹ w^67c23 flies. Nuclear extracts were prepared as previously described (Zhao et al. 1995 (link)) and immunoprecipitations were done using anti-FLAG M2 coupled to magnetic beads according to the manufacturer’s protocol (Sigma-Aldrich M8823). Two experimental and 3 control coimmunoprecipitation samples passed quality control (SDS-PAGE followed by Western blot for BEAF and silver stain for total protein) and were sent to the Thermo Fisher Scientific Center for Multiplexed Proteomics at Harvard Medical School for tandem mass spectrometry analysis. Differences between the experimental and control samples were evaluated for statistical significance by the Student’s t-test and corrected for multiple testing using the Benjamini–Hochberg correction. Results are in Supplementary Table 1.