Fig. 2: PKCλ/ι promotes EZH2 degradation through a ubiquitin-dependent mechanism.

a Immunoblots in HEK293T cells transfected with the indicated plasmids and quantification of HA-EZH2 (n = 3 independent experiments). b Immunoblots in LNCaP cells transfected with the indicated plasmids and treated with cycloheximide (CHX) (50 μg/ml) and MG132 (10 μM), Bafilomycin A1 (100 nM) or MLN4924 (1 μM) for 12 h, and quantification of HA-EZH2 (n = 3 independent experiments). c sgPRKCI and sgC LNCaP cells were incubated with 50 μg/ml of CHX at indicated time points, and quantification of EZH2 (n = 3 independent experiments). d Immunoblotting of EZH2 immunoprecipitates in sgPRKCI and sgC LNCaP cells, transfected with the indicated plasmids (n = 2 independent experiments). e Signal Intensity of EZH2-RBBP6 interaction measured by Mass Spectrometry in sgC and sgPRKCI HEK293T cells transfected with HA-EZH2 (n = 1 sample per condition). f Immunoblotting of HA-tagged immunoprecipitates of sgPRKCI and sgC LNCaP cells, transfected with the indicated plasmids (n = 2 independent experiments). g Immunoblotting of EZH2 immunoprecipitates in sgPRKCI and sgC LNCaP cells (n = 2 independent experiments). h Immunoblots in HEK293T cells, transfected with the indicated plasmids and quantification of EZH2 (n = 3 independent experiments). i Immunoblots in LNCaP cells, transduced with siRNAs and quantification of EZH2 (n = 3 independent experiments). j Immunofluorescence staining of EZH2 in LNCaP cells transduced with siRNAs and quantification of the EZH2 intensity (siC: n = 143, siRBBP6: n = 138 cells examined). Scale bars 10 μm. k Immunoblots in sgPRKCI and sgC LNCaP cells, transduced with the indicated siRNAs and quantification of EZH2 (n = 3 independent experiments). l Immunoblotting of EZH2 immunoprecipitates in LNCaP cells, transduced with the indicated siRNAs (n = 2 independent experiments). m LNCaP cells, transduced with the indicated siRNAs, were treated as in (c), and EZH2 quantification (n = 3 independent experiments). Immunoblot experiments were performed at least two times independently, with similar results. Data shown as mean ± SEM of 3 biological replicates (a, b, c, h, i, k, m). Two-tailed unpaired Student’s t-test (a, b, h, i, j, k). Two-way ANOVA (c, m). Source data are provided as a Source Data file.