Fig. 2
USP15 interacts with BARD1 BRCT domain through its C-terminal region. a Reciprocal endogenous immunoprecipitation (IP) between USP15 and BARD1 were performed in HEK293T cells. (Left panel) IP with anti-BARD1 antibody and blot with anti-BARD1 or USP15 antibody, respectively. (Right panel) IP with anti-USP15 antibody and blot with anti-USP15 or BARD1 antibody, respectively. b Co-immunoprecipitation (co-IP) assays were performed to check the interaction between USP15 and BARD1 upon DNA damage. HEK293T cells treated as indicated were lysed and immunoprecipitated with anti-BARD1 antibody, and Western blot was performed with indicated antibodies. c Schematic representation of USP15-truncated mutants used in this study (upper panel). Plasmids encoding HA-tagged full-length or deletion mutants of USP15 were co-transfected with plasmids encoding FLAG-tagged full-length BARD1 into 293T cells. Immunoprecipitation and immunoblotting were performed 48 h post transfection as indicated. d Schematic representation of BARD1-truncated mutants used in this study (upper panel). Plasmids encoding FLAG-tagged full-length or deletion mutants of BARD1 were co-transfected with plasmids encoding HA-tagged full-length USP15 into 293T cells. Immunoprecipitation and immunoblotting were performed 48 h post transfection as indicated. e USP15-knockout U2OS direct repeat green fluorescent protein (DR-GFP) cells were reconstituted with HA-USP15 wild-type (WT) or D3 mutant and homologous recombination (HR) efficiency were determined. Data are presented as mean±SD of three independent experiments. Two-tailed Student's t test, *P < 0.05, **P < 0.01. f The indicated cells“ response to poly-(ADP-ribose) polymerase (PARP) inhibitor (AZD2281) were measured by colony formation assay. Data are presented as mean ± SEM of three independent experiments, two-way analysis of variance (ANOVA), *P < 0.05. Unprocessed scans of blots are provided in Supplementary Fig. 8
