USP36 mediates EZH2 K222 deubiquitination and stabilizes EZH2. (A) USP36 overexpression led to the stabilization of exogenously overexpressed EZH2, which could be rescued by USP36 C131A mutant transfection in HEK293T cell. Cells were harvested for immunoblots 48 hours after transfection. (B) Overexpression of USP36 led to deubiquitination of EZH2 in HEK293T cells, and this is partially rescued by USP36 C131A mutant transfection. MG132 was given at 5 µM for 6 hours of treatment 48 hours after transfection. Densitometry analysis was used to quantify average changes in 3 individual experiments. (C) Knock-down of USP36 led to a decrease of EZH2 protein in KHYG-1 cells. The mRNA level of USP36 was indicated. Data are shown as mean ± SD, N = 3. *P < .05; **P < .01; ***P < .001. Cells were harvested for RNA extraction and immunoblots 24 hours after transfection. (D-E) Interaction between exogenously overexpressed EZH2 and USP36 wild-type or C131A mutant. Cells were harvested for co-IP 48 hours after transfection. (F) Change of interaction between USP36 and EZH2 wild-type or its mutants in HEK293T cells. Cells were harvested for co-IP 48 hours after transfection. All immunoblots were performed in at least 3 individual experiments; representative images are shown.