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.

Following processing and filtering, a high-confidence TF interaction map was generated using the HGSCore method to distinguish specific interactions as described previously (FBrf0216491), but with indirect prey-prey interactions filtered out to focus the network on the TF-interacting subspace. To draw the cutoff for interaction specificity and determine the FDR, we ran HGSCore on 40 simulated data sets randomly sampled from the real data set, until convergence on a cutoff score, resulting in a 2% FDR.