Fig. 9: UBE2K modulates ubiquitination and proteasomal degradation of histone H3.

a In vitro ubiquitination assay of 6xHis-tagged H3F3A with UBE2K and FLAG::RNF2 or GFP::RNF138 ubiquitin ligases followed by immunoblotting with antibodies to 6xHis, UBE2K, FLAG and GFP. The images are representative of two independent experiments. b In vitro ubiquitination of recombinant p53 followed by immunoblotting with antibodies to p53, UBE2K, and GFP. The images are representative of two independent experiments. c In vitro ubiquitination of 6xHis::H1 followed by immunoblotting with antibodies to 6xHis, UBE2K, and GFP. The images are representative of two independent experiments. d Western blot of UBE2K overexpressing (OE) HEK293 with antibodies to H3, H3K9me3 and β-actin. The graphs represent the relative percentage of H3/β-actin and H3K9me3/H3 to DMSO-empty vector cells (mean ± s.e.m. of four independent experiments). When indicated in the figure, cells were treated with 5 µM MG-132 for 16 h. e Knockdown levels of proteasome activators in HEK293 cells. The graph (relative expression to non-targeting (NT) shRNA HEK293 cells) represents the mean ± s.e.m. (PSMD11 shRNA (n = 8), PSME4 shRNA (n = 5), PSME3 shRNA (n = 6)). f Percentage of chymotrypsin-like proteasome activity relative to NT shRNA HEK293 cells (mean ± s.e.m. of three independent experiments). MG-132 treatment: 5 µM MG-132 for 16 h. g Western blot of HEK293 cells with antibodies to H3 and UBE2K. The graph represents the relative percentage of H3/β-actin to NT shRNA cells (mean ± s.e.m. of three independent experiments). h Western blot of HEK293 cells with antibodies to H3, PSMD11 and UBE2K. The graph represents the relative percentage of H3/β-actin to NT shRNA + empty vector cells (mean ± s.e.m. of three independent experiments). i After immunoprecipitation with anti-H3 and anti-FLAG antibodies in HEK293 cells, we performed a re-immunoprecipitation (Re-IP) with the same antibodies. Re-IP was followed by western blot with antibodies against H3 and polyubiquitinated proteins (polyUb) to detect immunoprecipitated H3 protein and polyUb-H3, respectively. The images are representative of two independent experiments. Prior to immunoprecipitation, cells were treated with 5 µM MG-132 (16 h). All the statistical comparisons were made by two-tailed Student’s t-test for unpaired samples. P value: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. NS not significant.