Figure 2.

TGF-β induces Smad3-dependent association of p130Cas and TβRI. (A) HEK293T cells transfected with the indicated plasmids were incubated with or without 2 ng/ml TGF-β, after which their lysates were immunoprecipitated with anti-p130Cas mAb and immunoblotted with anti-Flag mAb. (B) HaCaT cells were incubated with or without 2 ng/ml TGF-β, after which their lysates were immunoprecipitated with anti-Smad3N Ab and immunoblotted with anti-p130Cas mAb. (C) HEK293T cells transfected with Myc-Smad3 (WT) or a phosphorylation mutant (S3A or S3D), after which their lysates were immunoprecipitated with anti-Cas2 Ab and immunoblotted with anti-Myc mAb. Relative band intensities quantified by densitometry are indicated numerically below. (D) HEK293T cells were transfected with Myc-Smad3 and incubated with or without TGF-β for the indicated times. Cell lysates were immunoprecipitated with anti-Cas mAb and immunoblotted with anti-phospho-Smad3 Ab to detect p130Cas-bound phospho-Smad3. (E) Cas+/+ MEFs were incubated with or without 2 ng/ml TGF-β, after which their lysates were immunoprecipitated with anti-TβRI Ab and immunoblotted with anti-p130Cas mAb. (F) Smad3+/+ and Smad3−/− MEFs treated with 2 ng/ml TGF-β were subjected to immunoprecipitation as described in D.