Figure 4.

BARD1–BRCA1 damage-induced complexes are biochemically distinct. (A) eBARD1/BRCA1-containing complexes were purified from irradiated HeLa S3 nuclear extracts by Flag IP and elution with Flag peptide. Eluted material was then separated by centrifugation through a 10%–40% glycerol gradient, and alternate fractions (renumbered 1, 2, 3 etc.) were probed by Western blot with Abs to the proteins listed. (B) Flag-eluted material (input) was immunoprecipitated with either mouse IgG (negative control) or a mouse monoclonal Ab specific for Rad50 or for BACH1. Immunoprecipitated material was then immunoblotted with the indicated Abs. (C) HeLa cells were stably transduced with a retrovirus encoding an shRNA for Luciferase (Luc), for BACH1, or for CtIP. (Right) After 5 Gy, BRCA1 IP and Western blotting for BRCA1 and TopBP1 was performed. (Left) Standardized quantities of control whole-cell extract (WCE) were probed for BRCA1, BACH1, CtIP, and TopBP1. (D) One hour after 10 Gy IR, BRCA1 was immunoprecipitated from HCC1937 cells that had been reconstituted with vector or wild-type BRCA1. Western blotting for coimmunoprecipitated proteins was performed. (E) Myc-tagged wild-type and BACH1 mutant alleles were transfected into 293 cells and immunoprecipitated with anti-myc Ab 2 h after 10 Gy IR. Western blotting was performed for TopBP1 and Myc-tagged BACH1 (eBACH1).