Active site-targeted covalent irreversible inhibitors of USP7 impair the functions of Foxp3+ T-regulatory cells by promoting ubiquitination of Tip60
Fig 2
P217564 selectively inhibits USP7 activity in cultured cells.
(A) Jurkat cells were treated with DMSO or P217564 as indicated for 2 hours; USP7 was isolated by immunoprecipitation (IP) and its activity quantified by UB-EKL-based activity assay (middle). Simultaneously, equal amounts of crude cell extracts were used to determine total DUB activity using the UB-EKL assay (Right). (B) Schematic of Ub-VME reaction. (C) Jurkat cells were treated with DMSO or P217564; crude cell lysates were incubated with the Ub-VME probe and then immunoblotted against USP7 to determine formation of USP7-Ub-VME complexes. (D) Jurkat cells were treated with DMSO or P217564; crude cell extracts were labeled with HA-Ub-VME probe, followed by immunoblotting (IB) with indicated antibodies.