Fig. 4

Knockdown of Smad7 reverses inhibition of TGF-β/Smad3 signaling by YPA/TAZ silencing. a Reduction of elevated Smad7 by siRNA-mediated knockdown. Smad7 protein levels were determined by ProteinSimple capillary electrophoresis immunoassay. Data are representative of two independent experiments. b and c Restoration of TGF-β1-induced Smad3 phosphorylation by Smad7 knockdown. Fibroblasts were transfected with the indicated siRNAs and 48 h treated with TGF-β1 (5 ng/ml) for 1 h. Protein levels of total Smad3, phospho-Smad3, and beta-actin (loading control) were determined by ProteinSimple capillary electrophoresis immunoassay. N = 2, *p < 0.05 vs CTRL siRNA with TGF-β1, **p < 0.05 vs YAP/TAZ siRNA with TGF-β1. d Restoration of TGF-β/Smad3 activity by Smad7 knockdown. Fibroblasts were co-transfected with non-specific control or YAP/TAZ siRNAs (400 nM) and/or Smad7 siRNAs along with Smad Binding Element (SBE) luciferase reporter and β-galactosidase expression vector. 32 h later, cells were treated with vehicle (PBS) or TGF-β1 (5 ng/ml) for 16 h. SBE luciferase activities were determined 48 h after transfection and normalized to β-galactosidase activity. Bars indicate fold change in SBE reporter activity relative to control siRNA. N = 3, *p < 0.05 vs control siRNA with TGF-β1,**p < 0.05 vs YAP/TAZ siRNA with TGF-β1. All data are expressed as mean±SEM