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. 2023 Mar 11;42(17):1360–1373. doi: 10.1038/s41388-023-02631-8

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 5 — A Venn diagram depicting overlap of proteins co-immunoprecipitating with EZH2 from C006-M1, 28:B4:F3, IGR37, A375 and LM-MEL-45 cells (data derived from n = 3 biological replicates). B HA-tagged EZH2 and Flag-HA-tagged UBR4-LD (ligase domain) were co-expressed in HEK293 cells maintained ±MG132. Interactions between EZH2 and UBR4-LD were determined by immunoprecipitation with anti-EZH2 antibody and western blotting with anti-Flag antibody. C EZH2 protein was determined in HEK293 cells overexpressing either UBR4-LD or UBR4-FL (full length). UBR4 overexpression was measured by qPCR (lower panel: quantification relative to vector control, error bars: + SD). D UBR4 in 28:B4:F3, C006-M1 and HEK293 cells was knocked down by siRNA. EZH2 levels determined by western blot (top panels). Knockdown efficiency of UBR4 measured by qPCR as in (C). E Stability of EZH2 according to UBR4 levels (unaltered vs knockdown) was determined by western blot in A375 cells treated with cyclohexamide (CHX). Representative blot shown at the top, time-course plot at the bottom. F Ubiquitination of EZH2 was determined by anti-HA IP followed by western blot in A375 cells. G HEK293 cells with or without siUBR4 were transfected with HA-tagged EZH2 or V5-tagged UBR4-FL followed by 50 µM MG132 treatment for 4 h. Ubiquitination of EZH2 was determined by western blot with anti-ubiquitin (Ub) antibody. H B16-F10 cells ±siUBR4 were transfected with either Flag-tagged Ube2l6-WT (“wt”) or Flag-tagged Ube2l6-C87A (“mut”) and EZH2 levels determined by western blot. I–K To evaluate EZH2/UBR4/UBE2L6 interactions, B16-F10 cells ±siUBR4 were transfected with either Flag-tagged Ube2l6-WT (“wt”) or Flag-tagged Ube2l6-C87A (“mut”) followed by 50 µM MG132 treatment for 4 h and then: (I) EZH2, UBR4 and UBE2L6 protein estimation by western blot, (J) determination of ubiquitination of EZH2 by western blot with anti-Ub antibody, and (K) co-immunoprecipitation of UBR4 and UBE2L6 with EZH2 antibody. Data represent n = 3 biological replicates. Data for D and E are from three independent experiments and presented as means ± SD, analyzed by one-way ANOVA plus Tukey’s multiple comparison test. **p < 0.01.