Figure 5: Regulation of the DDR signalling by USP13.

(a) HEK293T cells were pretreated with DMSO or 25 μM Ku55933 for 2 h following which they were left untreated or treated with cisplatin. After an additional 1 h, USP13 was immunoprecipitated, left untreated or treated with phosphatase, and immunoblotted with phospho-SQ/TQ (pSQ/TQ) antibody. (b) HEK293T cells transfected with indicated constructs were left untreated or treated with cisplatin. HA-USP13 was immunoprecipitated and blotted with phospho-SQ/TQ (pSQ/TQ) antibody. (c) ATM-proficient cells (C3ABR) and ATM-deficient cells (L3) were treated as indicated. After 1 h, USP13 was immunoprecipitated and blots probed with the antibody specifically recognizing phosphorylation of USP13 at T196. (d) Co-localization of WT or T196A USP13 with γ-H2AX at DSB site created by I-SceI. Positive staining cells are quantified in the right panel. Scale bar, 5 μm. Error bars represent±s.e.m. from three independent experiments. >100 cells were counted per experiment. (e) Control, USP13 knockout, and USP13 knockout cells stably expressing the indicated constructs were subjected to colony formation assay to assess the sensitivity of cells to cisplatin. Error bars represent±s.e.m. from three independent experiments. (f) HEK293T cells transfected with HA-MDC1 were left untreated or treated with cisplatin. Cell lysates were subjected to immunoprecipitation with HA antibody. The immunoprecipitates were then blotted with the indicated antibodies. (g) HEK293T cells transfected with deletion mutants of HA-MDC1 were subjected to co-immunoprecipitation as in f. (h) HEK293T cells transfected with WT or T196A USP13 were treated with cisplatin, and cell lysates were incubated with Sepharose coupled with GST or GST-MDC1-FHA domain. After washing, proteins bound on Sepharose were blotted with the indicated antibodies. (i) Nonphosphorylated or phosphorylated Thr 196 peptide (T196 or p-T196, respectively) was conjugated to Sepharose beads and incubated with purified GST-MDC1-FHA domain in NETN buffer. After washing, proteins bound to beads were blotted with the indicated antibodies.