Fig 3. vFLIP and vCyclin decrease SMAD2 protein expression and desensitize TGF-β signaling.

(A) SMAD2 expression was drastically decreased upon the expression of vFLIP or vCyclin in SLK cells. Western blot for SMAD2 was performed with SLK cells transfected with empty vector or with vFLIP and vCyclin expression vectors. Actin was detected as internal control. (B and C) vFLIP (B) and vCyclin(C) diminished the response of SLK cells to TGF-β ligand. Luciferase reporter analysis was carried out with pGL3-SBE4, which contains 4 SMAD-binding elements, co-transfected with empty vector or with vectors expressing vFLIP or vCyclin in the absence or presence of TGF-β ligand. TGF-β ligand was added 24 hrs after transfection and the cells were harvested 72 hrs after transfection. The luciferase activities in the presence of TGF-β were normalized with the activities in the absence of TGF-β.