IndraLab

"After 1 h, USP13 was immunoprecipitated and blots probed with the antibody specifically recognizing phosphorylation of USP13 at T196."

"(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."