Figure 3. USP7 deubiquitinates PHF8.

(A) MCF-7 cells with Dox-inducible expression of FLAG-USP7/WT were cultured in the absence or presence of increasing amounts of Dox. Cellular extracts and total RNA were collected for Western blotting (left panel) and qRT-PCR (right panel) analysis, respectively. (B) Experiments analogous to A were performed in U2OS cells with Dox-inducible expression of FLAG-USP7/C223S. (C) MCF-7 cells were cultured in the absence or presence of increasing amounts of HBX 41,108 for 2 hours as indicated. Cellular extracts and total RNA were collected for Western blotting (left panel) and qRT-PCR (right panel) analysis, respectively. (D) HeLa cells stably expressing FLAG-PHF8 were cotransfected with different amounts of Myc-USP7 and HA-Ub/WT or HA-Ub/mt as indicated. Cellular extracts were immunoprecipitated with anti-FLAG followed by IB with anti-HA. (E) HeLa cells stably expressing Myc-PHF8 were cotransfected with HA-Ub/WT and FLAG-USP7/WT or USP7/ΔMATH. Cellular extracts were immunoprecipitated with anti-Myc followed by IB with anti-HA. (F) HeLa cells (left panel) or MCF-7 cells (right panel) stably expressing FLAG-PHF8 were cotransfected with HA-Ub/WT and control siRNA or USP7 siRNAs as indicated. Cellular extracts were immunoprecipitated with anti-FLAG followed by IB with anti-HA. (G) MCF-7 cells stably expressing FLAG-PHF8 were transfected with HA-Ub/WT and cultured in the presence or absence of HBX 41,108. Cellular extracts were immunoprecipitated with anti-FLAG followed by IB with anti-HA. (H) In vitro deubiquitination assays with HA-Ub–conjugated PHF8 purified from HeLa cells using high-salt buffer and USP7/WT or USP7/C223S purified from extracts of baculovirus-infected insect cells. In A–C, each bar represents the mean ± SD for biological triplicate experiments. **P < 0.01, 1-way ANOVA.