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. 2021 Apr 13;49(8):4421–4440. doi: 10.1093/nar/gkab209

Figure 2.

Figure 2.

USP7 promotes deubiquitination and stabilization of EZH2. (A) A375 cells transfected with control siRNA or different sets of USP7 siRNAs were collected and analyzed by western blotting and quantitative reverse transcription (qRT)-PCR. Each bar represents the mean ± SD from biological triplicate experiments. **P < 0.01, by one-way ANOVA. (B) A375 cells transfected with Control siRNA or USP7 siRNAs were treated with DMSO or proteasome inhibitor MG132 (10 μM). Cellular extracts were prepared and analyzed by western blotting. (C) A375 cells were transfected with Control siRNA or USP7 siRNA followed by treatment with cycloheximide (CHX, 50 μg/ml), and harvested at the indicated time points followed by western blotting analysis. (D) A375 cells with Dox-inducible expression of FLAG-USP7/WT or FLAG-USP7/C223S cultured in the absence or presence of increasing amounts of Dox. Cellular extracts and total RNA were collected for western blotting and qRT-PCR analysis, respectively. Each bar represents the mean ± SD from biological triplicate experiments. **P < 0.01, one-way ANOVA. (E) A375 cells stably expressing FLAG-EZH2 co-transfected with Control siRNA or USP7 siRNAs and HA-Ub/WT. Cellular extracts were immunoprecipitated with anti-FLAG followed by immunoblotting with anti-HA. (F) A375 cells stably expressing FLAG-EZH2 were co-transfected with HA-Ub/WT or HA-Ub/MT and different amounts of Myc-USP7 or Myc-USP7/C223S as indicated. Cellular extracts were immunoprecipitated with anti-FLAG followed by immunoblotting with anti-HA. (G) In vitro deubiquitination assays were performed with HA-Ub-conjugated EZH2 purified from A375 cells using high-salt and detergent buffer and USP7/WT or USP7/C223S purified from baculovirus-infected insect cells. The asterisks indicate the recombinant proteins stained by Commassie Blue.