Impaired Smad7-Smurf–mediated negative regulation of TGF-β signaling in scleroderma fibroblasts
J. Clin. Invest. Yoshihide Asano, et al. 113:253 doi:10.1172/JCI16269 [Go to this article.]

Figure 7
Constitutive complex formation of Smad7 with TβRI in scleroderma fibroblasts. (a) In the left panels, confluent quiescent normal fibroblasts were incubated in the absence or presence of TGF-β1 (2 ng/ml) for 24 hours. In the right panels, normal fibroblasts were transfected with the Smad7 expression vector (2 μg) and incubated for 48 hours prior to cell extraction. Some transfectants were stimulated with TGF-β1 (2 ng/ml) for the last 3 hours. Cell lysates (1 mg of protein/sample) were subjected to immunoprecipitation (IP) using anti-TβRI Ab. Immunoprecipitates were subjected to immunoblotting (Blot) using anti-Smad7 Ab. After stripping, total TβRI levels were determined by immunoblotting. Total Smad7 levels were determined by immunoblotting using whole-cell lysates (20 μg of protein/sample). (b) Cell lysates prepared from confluent quiescent fibroblasts were subjected to immunoprecipitation and immunoblotting as described above (upper panels). Reverse immunoprecipitation using anti-Smad7 Ab was also performed (lower panels). Each figure shows one representative of five independent experiments.