Figure 3.

USP7 deubiquitinates Chk1 and itself in vivo. (A) In vivo Chk1 ubiquitination. 293T cells were transfected with EV or His-Ub, alone or together with Flag-USP7 WT. Cells were incubated for 16 h with MG132 before lysis. Then a pull down was performed with Ni-NTA beads followed by western blot analysis with the indicated antibodies. (B) In vitro Chk1 deubiquitination assay. 293T cells were transfected with EV or His-Ub. Cells were incubated for 16 h with MG132, lysed and a Ni-NTA pull down was performed. Subsequently, beads were washed and incubated with purified USP7 WT or CI, in the absence or presence of NEM. (C) 293T cells were transfected with USP7 WT or CI, together with His-Ub. Cells were lysed and an anti-Flag immunoprecipitation was performed followed by western blot analysis with the indicated antibodies.