Each purified bait clone (DPiM_cDNA_bait_cDNA) was used to transiently transfect S2R+ cells in a 54ml culture. The individual S2R+ cultures were induced to produce FLAG-HA fusion protein by treatment with media containing 0.35 mM Cu2SO4 for 20 hours. Cells were lysed in buffer containing protease inhibitor and passed through a 0.45um durapore filter.

Each clarified lysate was bound overnight to crosslinked HA immunoaffinity resin. Unbound proteins were washed off with lysis buffer followed by PBS and then bound protein complexes were competitively eluted using synthetic HA peptide YPYDVPDYA (250 μg/ml) in PBS.

Copurified proteins were precipitated using trichloroacetic acid, washed with acetone, dried, digested overnight with trypsin, and analyzed by LC-MS/MS. LC-MS/MS spectral data were searched with SEQUEST (Eng et al., 2008) against a database of D. melanogaster proteins derived from FlyBase version 5.23. The LC-MS/MS identifications were filtered to, on average, a 0.3% peptide FDR.

The network of protein interactions filtered to 5% FDR was clustered using MCL (Enright et al., 2002) (inflation coefficient of 3.0 and pre-inflation factor of 0.01). Resulting p values were further filtered to adjust for multiple hypothesis testing, keeping clusters based on a 0.01 Q-value cutoff (Storey and Tibshirani, 2003).