Fig 3.

Cav-1 promotes Alk1/Smad1 signaling and is required for the TGFβ induction of Smad1 phosphorylation in dermal fibroblasts. (A) Human dermal fibroblast cultures isolated from normal (NS) and scleroderma (SSc) patients were treated with scrambled siRNA (scr) or siRNA targeted against cav-1 (cav-1 si) as described, and protein levels of cav-1 (cav-1), Smad1, phospho-Smad1, and β–actin were determined by Western blot. (B) Normal human dermal fibroblast cultures were treated with scrambled siRNA or siRNA targeted against cav-1, serum starved for 24 hrs, and treated with or without TGFβ for 30 min prior to collection of cell lysates. Levels of cav-1, phospho-Smad1, and β-actin protein were detected by Western blot. Basal P-Smad1 levels were detectable only after longer exposure compared to levels after TGFβ treatment. (C) Human dermal fibroblasts were grown to confluence and transduced with either cav1-Ad or control adenovirus (LucAd) for 24 hrs, followed by serum starvation for 24 hrs. A dose response of cav1-Ad was performed and a dose-dependent increase in phosphorylated Smad1 was detected by Western blot.