FIG. 5.

Smurf-binding-deficient Smad7(ΔPY) is localized in the nucleus and retains the ability to inhibit TGF-β signaling. (A) TGF-β induces the nuclear export of Smad7(WT) but not Smad7(ΔPY) in COS7 cells. Cells were transfected with Flag-Smad7(WT) and Flag-Smad7(ΔPY). At 40 h posttransfection, cells were treated with 100 pM TGF-β1 for 45 min or left untreated. Smad7 subcellular localization was detected by indirect immunofluorescence using anti-Flag antibody, followed by FITC-conjugated secondary antibody, and visualized under a fluorescence microscope. Scale bar, 20 μm. (B) Smad7(ΔPY) suppressed CAGA-luciferase expression induced by TGF-β1. COS7 cells were transfected with CAGA-Luc (0.5 μg), Smad7(WT), and Smad7(ΔPY) (0.1, 0.5, and 1 μg, respectively) as indicated. At 24 h posttransfection, the cells were treated with TGF-β1 (50 pM) for 20 h or left untreated and then harvested for luciferase assay. The experiment was performed in triplicate, and the data represent the means and standard deviations of three independent experiments after normalization to Renilla luciferase activity. (C) Smad7(ΔPY) suppressed ARE-luciferase expression induced by TGF-β1. COS7 cells were transfected with ARE-Luc (0.5 μg) and with Smad7(WT) and Smad7(ΔPY) (0.1 and 0.5 μg, respectively) as indicated. The reporter assay was performed similarly to that in panel B.