Fig. 3

USP15 deubiquitinates BARD1 and facilitates its interaction with HP1γ. a Wild-type (WT) or USP15-knockout 293T cells were transiently transfected with indicated plasmids and treated with or without mitomycin C (MMC) for 24 h. Cells were lysed, and ubiquitinated BARD1 proteins were pulled down and were detected by Western blot. b USP15 deubiquitinates BARD1 BRCT domain in vitro. BARD1-BRCT conjugated with indicated ubiquitin chains were purified from 293T cells and were used as substrates, glutathione S-transferase (GST)-USP15 WT or GST-USP15 C269A were purified from E. coli, and then the in vitro deubiquitination assay were performed, as described in the Methods section. Samples were separated by sodium dodecyl sulfate (SDS) gel and blotted with indicated antibodies. c USP15 deubiquitinates BARD1 BRCT domain upon DNA damage. WT or USP15-knockout 293T cells were transfected with the indicated plasmids and treated with or without MMC for 24 h. Cells were lysed and ubiquitinated proteins were pull down by Ni-NTA. Samples were detected by Western blot with indicated antibodies. d USP15-knockout 293T cells reconstituted with HA-USP15 WT or C269A mutant were treated as indicated for 24 h, cells were then lysed, and ubiquitinated proteins were pull down by Ni-NTA. Samples were detected by Western blot with indicated antibodies. e USP15 facilitates BARD1’s interaction with HP1γ. The 293T cells were transfected with the indicated plasmids and treated with or without ionizing radiation (IR) (10Gy). Cell lysates were immunoprecipitated with FLAG M2 beads and subjected to immunoblot with indicated antibodies. f USP15-depleted 293T cells reconstituted with HA-USP15 WT or C269A mutant were treated with or without IR (10Gy). Cell lysates were immunoprecipitated with FLAG M2 beads and subjected to immunoblot with the indicated antibodies. g USP15-mediated BARD1-BRCT deubiquitination promotes BARD1–HP1γ interaction in vitro. (Upper panel) Workflow of the in vitro binding assay. Briefly, ubiquitinated BARD1-BRCT were purified from 293T cells by tandem immunoprecipitation (IP) (first HA beads then FLAG beads) and were left on FLAG beads. Then one-half part was left untreated and one-half part was deubiquitinated by His-USP15 in vitro. Next, the immunoprecipitates were incubated with glutathione S-transferase (GST) or GST-HP1γ in vitro. (Lower panel) Samples were detected by Western blot with indicated antibodies. Unprocessed scans of blots are provided in Supplementary Figs 8 and 9