Figure 2. EZH2 is stabilized through USP7-mediated deubiquitination. (A) HA-EZH2 was expressed in HEK293T cells with Flag-USP7 or Flag-USP7 C223S. Cells were treated with DMSO or 10 μM MG132 for 12 h before harvesting. Cell lysates were immunoblotted with anti-HA or anti-Flag antibody. (B) HEK293T cells were transfected with increasing amounts of Flag-USP7 or Flag-USP7 C223S. Cell lysates were immunoblotted to detect Flag-tagged USP7, EZH2, or H3K27me3. (C) HEK293T cells were transfected with increasing amounts of USP7 shRNAs. Cell lysates were immunoblotted to detect EZH2, USP7, or H3K27me3. (D) HEK293T cells expressing empty vector (control) or Flag-USP7/USP7 C223S were treated with cycloheximide (CHX) for the indicated durations. Cell lysates were immunoblotted with anti-EZH2 or anti-Flag antibody. The line graph represents the relative protein levels of EZH2 normalized to those of β-actin using a densitometer and expressed as relative intensity compared to the non-treated control. (E) DU145 or PC3 cells were treated with P5091 (12.5 μM) for 24 h and MG132 (10 μM) for 12 h. Cell lysates were immunoblotted to detect EZH2, USP7, or H3K27me3. (F), (G), and (H) HEK293T cells were transfected as indicated and treated with MG132 for 12 h before harvesting. Cell lysates were then subjected to immunoprecipitation with HA antibody followed by immunoblotting with anti-HA or anti-Xpress antibody.