Fig. 1.

RAP80 interacts with the BRCA1 BRCT motif and responds to IR independently of BRCA1. (A) Co-immunoprecipitation (IP) between endogenous BRCA1 and RAP80. (B) Lysates were prepared from 293T cells transfected with WT or clinical mutant myc-BRCA1-BRCT domains. Myc antibody immunoprecipitated material was separated by SDS–polyacrylamide gel electrophoresis, and immunoblotting (IB) was performed. (C) HeLa S3 cells stably expressing epitope tagged-WT or S101A mutant RAP80 were IR-treated, and lysates were probed with an antibody specific to phosphorylated (P) RAP80-S-101. E, Glu; L, Leu; N, Asn; R, Arg; and V, Val. (D) ATM −/− and +/+ and (E) HCC1937 cells were gamma irradiated, and IB was performed on cell lysates as indicated. (F) HCC1937 cells reconstituted with vector or WT BRCA1 were transfected with GFP-RAP80 and gamma-irradiated, and, 1 hour later, IF was performed to assess colocalization with γH2AX.