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. 2024 Mar 9;15:2163. doi: 10.1038/s41467-024-46422-5

Fig. 4. Mi-2β promotes the K510 methylation of EZH2 to inhibit the transcription of ISGs.

Fig. 4

a Mass spectral peptide count of Mi-2β-interacting proteins. b Endogenous Mi-2β-EZH2 interactions were detected by immunoprecipitation in B16F10 and the primary mouse melanoma cells (PM) (n = 3, independent experiments). c The amount of secreted Cxcl10 was measured in B16F10 cells with EZH2 silencing by ELISA (n = 3). d The epigenetic modifications of H3 (H3K27ac, H3K27me1 and H3K27me3) were measured in B16F10 cells or PM cells with EZH2 silencing by Western blot (n = 3, independent experiments). e ChIP–quantitative polymerase chain reaction for enrichment of H3K27me3/H3K27me1 at gene locus in B16F10 cells with shMi-2β (n = 3). f, g Mapping the interaction interface of EZH2 with Mi-2β by immunoprecipitation. HA-tagged EZH2 WT or deletion mutants and Flag-tagged Mi-2β were used as indicated and detected by immunoprecipitation in B16F10 cell. SANT1, SANT domain I; SANT2, SANT domain II; CXC, cysteine-rich domain; SET, methyltransferase catalytic domain (g: n = 3, independent experiments). h EZH2 lysine methylation was detected by immunoprecipitation with specific anti-Mi-2β antibodies, anti-EZH2 antibodies or anti- pan-monomethylated lysine antibodies, respectively (n = 3, independent experiments). Mi-2β (i) levels or total Mi-2β-Flag (j) in the whole-cell lysate (WCL) and pan-methylated lysine levels of immunoprecipitated EZH2, EZH2 K735A or EZH2 K510A were detected in B16F10 melanoma cells. Immunoglobulin G (IgG) Immunoprecipitation served as a negative control (n = 3, independent experiments). k The amount of secreted Cxcl9 and Cxcl10 were measured by ELISA in B16F10 cells of stable EZH2 silencing and EZH2 WT or mutant EZH2 reintroduction (n = 3). c, e, k Values represent mean ± SD. The unpaired, two tailed t-test. Source data are provided as a Source Data file.