Open reading frames were transferred from the BDGP Drosophila melanogaster expression-ready clone set (FBrf0213191) to the pMK33-C-FLAG-HA acceptor vector (see DPiM_cDNA_bait collection report). Each clone was transiently transfected into a 54 ml culture of Drosophila S2R+ cells. Protein expression was induced with 0.35 mM CuSO4 for 24 hours and nuclear extracts were prepared (PMID: 6828386) then diluted 1:1 with dialysis buffer (20mM HEPES [pH 7.6], 20% glycerol, 100 mM KCl, 2mM MgCl2, 0.1 mM EDTA, 1 mM DTT, 0.25 mM PMSF and Roche Complete protease inhibitor).

Each clarified lysate was bound to 40 ul of crosslinked HA immunoaffinity resin (Sigma) for 3 hr at 4oC. Unbound proteins were washed off with dialysis buffer. Bound proteins were eluted using IgG elution buffer (Thermo Scientific Pierce), and neutralized with 1 M Tris [pH 8.0].

The copurified proteins were precipitated using trichloroacetic acid, washed with TCA and acetone, dried, digested overnight with trypsin, and analyzed by LC-MS/MS. MS/MS spectra were searched with SEQUEST (PMID: 18774840) against FlyBase Release 5.41.

MS/MS spectra were searched with SEQUEST (PMID: 18774840) against FlyBase Release 5.41 and filtered to a 2.27% protein FDR for the entire data set with the reverse database approach (PMID: 17327847). Column carry-over between experiments was corrected with a statistical approach, incorporating peptide abundance and the probability of consecutive observations.