Active site-targeted covalent irreversible inhibitors of USP7 impair the functions of Foxp3+ T-regulatory cells by promoting ubiquitination of Tip60
Fig 8
P217564 inactivates USP7, resulting in increased ubiquitination and degradation of Tip60 and Foxp3 in Treg cells.
(A) Treg cells were isolated from mice and treated with 10 μM P217564 for 2 hours; crude cell extracts were incubated with Ub-VME probe, followed by IB against USP7 to analyze the formation of USP7-Ub-VME complexes. (B) Mouse Treg cells were treated with DMSO or 10 μM P217564 for 2 hours; crude cell extracts were subjected to SDS-PAGE electrophoresis, followed by IB with indicated antibodies. Relative quantitation of band intensity is shown. (C) P217564-induced ubiquitination of Tip60, Foxp3 and USP7 in Treg cells was analyzed using the DUB-based UbiTest described in Fig 6.