MELK mediates ubiquitination of EZH2. (A) Change of EZH2 ubiquitination level with MELK overexpression in HEK293T cells. Before cell harvesting, MG132 was given at 10 µM for 6 hours of treatment 48 hours after transfection. (B) Change of EZH2 ubiquitination level with MELK knock-down in NKYS cells. Forty-eight hours after transfection, 1 µM of MG132 was used for treatment of 6 hours before cell harvesting. (C) Change of EZH2 ubiquitination level with OTSSP167 treatment in NKYS cells. A total of 10 nM of OTSSP167 and 2 µM of MG132 was used for 6 hours’ treatment before harvesting. Densitometry analysis was used to quantify average changes in 3 individual experiments. (D) Schematic protocol of SILAC-MS experiment. (E) Expression level of HA-tagged EZH2 wild-type and ubiquitination-dead mutants in NKTL cell lines. The transfection was performed using electroporation. Cells were harvested for immunoblots 24 hours after transfection. (F) IP showing the change of ubiquitination level for EZH2 wild-type or mutants in HEK293T cells. Before cell harvesting, 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. (G) Change of interaction between MELK 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 and representative images are shown. CON, control; EV, empty vector; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HA, hemagglutinin; LC, liquid chromatography; Ub, ubiquitination; WT, wild-type.