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Figure 5


Fig. 5. Effect of selective downregulation of ALK1 and ALK5 receptors and Smad1 and Smad3 isoforms by siRNA on TGF-beta-induced ET-1 expression. (A) Efficiency of endogenous protein knockdowns by siRNA was confirmed by western blot assays. Functional validation of ALK1, ALK5, Smad1 and Smad3 siRNA was done by cotransfection with ALK1- and ALK5-specific reporters under overexpression of ALK1 constitutively active (ca) (B) and ALK5 ca (C) forms. Results are compared with the effect of an siRNA control. TGF-beta-induced ET-1 expression under specific downregulation of ALK1 and ALK5 receptors and Smad1 and Smad3 isoforms was analyzed at the level of ET-1 promoter activity (D) and peptide secretion (E), as previously described. Values are expressed as fold induction with respect to siRNA control with pCMV5 empty vector (B,C) or without TGF-beta (D,E) (mean ± s.d., n=3, *P<0.05 versus control without activation, #P<0.05 versus control with activation).