Direct BRCA1–ERM interaction detected by ligand overlay and coimmunoprecipitation. (A and B) HeLa P2 fractions were resolved by 1D SDS-PAGE (A) or 2D electrophoresis (B), blotted, and probed with EGFP-CTD detected with anti-EGFP antibody. (A) A 75–80-kD doublet was reproducibly detected (lane 3), whereas a third higher molecular mass band was not (lane 1). No signal was detected in a parallel blot using the control HeLa lysate containing EGFP-NTA (lane 2). The 75–80-kD doublet (lane 4) was excised, trypsin digested, and sequenced by LC-MS/MS (Table S1). The red 1 and 2 indicate the 80- and 75-kD bands, respectively. (B) 2D gel electrophoresis: multiple spots were detected in the 75–80-kD region. (a) SYPRO ruby staining; (b) Western blot; (c) detail of SYPRO ruby spots (1–8) cut out for LC-MS/MS analysis (Table S2, showing results for each spot). (C) Coimmunoprecipitation of ezrin with BRCA1. (top, 1) Lysate + beads + mouse anti-BRCA1 Ab1 (+), lysate + beads with no antibody (−), and whole cell lysate (WCL) probed with mouse anti-BRCA1 Ab1; (2) lysate + beads + mouse antitryptase (+), lysate + beads with no antibody (−), and whole cell lysate probed with mouse antitryptase; (3) beads + mouse IgG1 probed with anti–mouse IgG1. (bottom) The same samples as in the top immunoblotted for ezrin. Ezrin protein was only detected in the BRCA1 sample (4) when Ab1 was coupled to the beads (+). Beads with no antibody (−) and both the tryptase and mouse IgG1 control samples (5 and 6, respectively) were negative for ezrin. In replicate experiments, ezrin was reproducibly detected (mean band intensity in the BRCA1 pull-down 20.0 [experiment 1] or 29.9 [experiment 2] vs. intensity in control pull-down 3.25 [experiment 1] or 1.03 [experiment 2]).