Figure 8.

The effect of EZH2 on PAR₄-activated β-catenin half-life. (A) Western blot analysis of β-catenin in HEK293 cells co-transfected with 0.4 μg hPar4, 0.3 μg flag-β-catenin and 0.5 μg ezh2-HA plasmids and activated by PAR₄ with 200 µM AYPGKF for 2 h followed by cycloheximide treatment (100 μg/mL) for between 5 min and 2 h. Western blot analysis was performed on cell lysates to detect β-catenin using anti-flag antibody (1:1000) and normalized to β-actin (1:1000) for protein loading. (B) 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 and treatment with cycloheximide, lysates were collected, and luciferase activity was normalized to β-gal activity to control for transfection efficiency. This experiment was carried out in triplicate.