Figure 6.

PAR₄ (A) and PAR₂ (C) induced EZH2 binding to β-catenin and lysine (K) methylation. (A,B) HEK293 cells were co-transfected with 1.2 μg Par4 (A) or Par2 (B), 2.4 μg HA-ezh2 and 1.5 μg flag-β-catenin. Cell lysates were immunoprecipitated (600 mg per assay) following 200 μM AYPGKF activation (A) or 200 μM SLIGKV activation (B) for between 15 min and 4 h using anti-HA antibody (3 mL antibody per assay). Detection by Western blot analysis of β-catenin-EZH2 association was performed using anti-flag antibody (1:1000 dilution) for β-catenin. Methylation was detected using anti-Methyl K antibody (1:500 dilution). EZH2 levels were evaluated in total cell lysate for normalization (1:500 dilution). Each experiment was performed twice. (B,D) Quantification of bands was carried out using Image J software. (E) The effect of EZH2 on PAR-activated LEF/TCF transcriptional activity. (A) PAR₄ induced Lef/Tcf in the presence of EZH2. HEK293 cells were co-transfected with 0.075 μg TOPflash, 0.125 μg LEF, 0.2 μg β-gal, 0.2 μg Par4 and 0.1 μg ezh2-HA. Following activation with 200 μM AYPGKF, lysates were collected, and luciferase activity was normalized to β-gal activity to control for transfection efficiency. (F) PAR₂ induced Lef/Tcf in the presence of EZH2. HEK293 cells were co-transfected with 0.075 μg TOPflah, 0.125 μg LEF, 0.2 μg β-gal, 0.3 μg Par2 and 0.3 μg ezh2-HA. Following activation with 200μM SLIGKV, lysates were collected, and luciferase activity was normalized to β-gal activity to control for transfection efficiency. These results are representative of the experiment performed in quadruplicate. The results are the mean of duplicates in each experiment. In all our Lef/Tcf experiments, we compared two groups of equally loaded plasmids. The total load of the plasmids did not exceed ~1.1 mg.