Figure 5. ETV4 interacts with COP1 to regulate nuclear β-catenin stability.

(A) GIST T1 cells were transfected with either ETV4 siRNA or control siRNA for 72h, and then cells were treated with MG132 10 μM for 6h prior to harvest. Nuclear protein extracts were analyzed by western blot as indicated. (B) GIST T1 cells were transfected with control or human ETV4 plasmids for 48h and treated with MG132 10 μM overnight before harvesting. Nuclear protein extracts were analyzed by western blot as indicated. (C) Representative CHX-chase assays (of 2 performed) to determine the stability (half-life) of nuclear β-catenin in GIST T1 cells 48h after transfection with control siRNA or ETV4 siRNA. Cells were collected after the addition of 200 mg/ml CHX at the indicated time points. Relative nuclear β-catenin levels were determined by normalizing to the loading control (lamin B1) and then normalizing to the t = 0h control siRNA. Immunoblots of nuclear extracts are shown. (D) GIST T1 cells were transfected with the indicated siRNA and harvested 48h later and nuclear extracts were analyzed by western blot. (E) GIST T1 cells were transfected with control or ETV4 siRNA for 72h and then treated with MG132 10 μM for 6h prior to harvest. Nuclear extracts were immunoprecipitated by either anti-β-catenin or anti-COP1 and western blot was performed as indicated. (F) GIST T1 cells were transfected with the indicated constructs and harvested 48h later and nuclear extracts were analyzed by western blot.