Fig. 3: HspBP1 interacts with BRCA1 and is recruited to sites of DSBs.

A HeLa cells were either untreated or treated with 5 Gy of IR for 3 h, after which whole-cell lysates were subjected to immunoprecipitation with anti-HspBP1 followed by western blotting with the indicated antibodies. B HeLa cells were prepared as in (A), and lysates were subjected to immunoprecipitation with anti-BRCA1 antibody followed by western blotting with the indicated antibodies. C HCC1937 and HCC1937-BRCA1 cells were treated with or without 5 Gy of IR. 3 h after IR treatment, whole-cell lysates were immunoprecipitated with anti-HspBP1 or anit-BRCA1 antibodies and subjected to western blot analysis with indicated antibodies. D HA-tagged BRCA1 was co-transfected with GFP-tagged HspBP1 into HEK293T cells and subjected to 5 Gy of IR treatment for 3 h, after which whole-cell lysates were subjected to immunoprecipitation with anti-HA antibody followed by western blotting with the indicated antibodies. E HeLa cells were treated with or without 5 Gy of IR. 3 h after IR treatment, lysates of the cytosolic faction and nuclear faction were immunoprecipitated with anti-HspBP1 antibody and subjected to western blot analysis with indicated antibodies. F HeLa cells were either untreated or treated with 5 Gy of IR and fixed at 3 h after irradiation. Localization of endogenous HspBP1 and γ-H2AX was detected using antibodies against HspBP1 (red) and γ-H2AX (green), and colocalization appears yellow in the merged image. Nuclei were stained with DAPI. Representative images and the percent colocalization between HspBP1 and γ-H2AX is shown in the graph. At least 100 cells were analyzed for each time point and data are presented as mean ± SD (n = 3), **P < 0.01, two-tailed Student’s t-test.