MELK affects EZH2 ubiquitination following site-specific phosphorylation. (A) Schematic model showing localizations of S220 phosphorylation site and K222 ubiquitination site on EZH2 protein. (B) Amino acid sequence of E1 and p-E1 peptides. (C) In vitro kinase assay showing MELK could phosphorylate EZH2 at S220. (D-E) Different ratio of cell survival for (D) NKYS and (E) KHYG-1 cells transfected with empty vector, EZH2 wild-type or mutant plasmids. NKYS and KHYG-1 cells were transfected with indicated plasmids through electroporation and subject to overnight puromycin selection (1 µg/mL) 7 hours after transfection. Cell survival was measured using CellTiter-Glo reagent. (F) Change of interaction between MELK and EZH2 wild-type or mutants in HEK293T cells. Cells were harvested for co-IP 48 hours after transfection. Densitometry analysis was used to quantify average changes in 3 individual experiments. (G) Schematic diagram indicating the conservation of EZH2 S220 and K222. (H-I) Change of EZH2 total serine phosphorylation upon 16 hours of OTSSP167 treatment in NKYS (H, 10 nM) and NK-S1 (I, 20 nM) cells. All immunoblots were performed in at least 3 individual experiments; representative images are shown.