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. 2020 Sep 7;27(12):3226–3242. doi: 10.1038/s41418-020-00615-9

Fig. 4. PRMT1-mediated meR342-EZH2 prevents TRAF6-mediated EZH2 ubiquitination and degradation.

Fig. 4

a EZH2 protein contains 3 potential TRAF6-binding sites. The consensus TRAF6-binding site motif has been described previously. b, c Western blot analysis of endogenous and exogenous interaction between EZH2 and TRAF6 after IP EZH2 in MM-231 cells (b) and HEK293T cells (c), respectively. d FLAG-tagged TRAF6 plasmids were transfected into 293T cells, and FLAG beads were used for IP. Precipitates were subjected to western blot analysis with indicated antibodies. e, f Detecting Flag-EZH2-associated ubiquitination by immunoblotting after IP HA-Ub in HEK293T cells transfected with indicated constructs. g Detecting Flag-EZH2-associated K48 and K63 ubiquitination by western blot after IP HA-Ub in MM-231-shEZH2 cells transfected with indicated constructs. h Immunoblotting analysis the amount of TRAF6 binding with EZH2 in MM-231-shCtrl and MM-231-shPRMT1 cells after IP EZH2. i Western blot detection of exogenous Flag-EZH2 binding with TRAF6 after IP Flag in Flag-EZH2-WT overexpression or Flag-EZH2-WT and His-PRMT1 co-expression HEK293T cells. j IF analysis of EZH2 and TRAF6 in MM-231-shEZH2-WT cells and MM-231-shEZH2-R342K cells. k Detecting the level of Flag-EZH2 interacting with TRAF6 after IP Flag in Flag-EZH2-WT or Flag-EZH2-R342K overexpression cells. l, m Western blotting analysis of Flag-EZH2-R342K binding with TRAF6 (l) and Flag-EZH2-R342K ubiquitination (m) after IP Flag in MM-231-shEZH2-R342K cells treated with CDK1 inhibitor CGP.