β-Catenin–p-Smad2 complex formation requires wt α3 and is essential for TGF-β1–mediated up-regulation of mesenchymal genes. (A) β-Catenin effectively immunoprecipitates p-Smad2 in α3 wt cells but not in α3^−/− or H245A mutant cells after TGF-β1 stimulation. (B) Inhibition of β-catenin by expression of a dominant-negative β-catenin (dnβ-cat; left) or an shRNA against β-catenin (shβ-cat; right) in α3 wt cells inhibits TGF-β1–induced α-SMA but not Smad2 phosphorylation compared with wt α3 cells transfected with vector alone, nonsilencing control shRNA (shctl), or left nontransfected. (C) Tyrosine phosphorylation of β-catenin and formation of the pY654–β-catenin–p-Smad2 complex is only detected in α3 wt cells treated with TGF-β1 but not in α3^−/− or H245A mutant cells. (D) Tyrosine phosphorylation of β-catenin (β-cat) and formation of the pY654–β-catenin–p-Smad2 complex in α3 wt cells requires endocytosis. α3 wt cells untreated or exposed to 300 µM of the clathrin inhibitor MDC were stimulated with TGF-β1 and analyzed for pY654–β-catenin–p-Smad2 complexes by co-IP. The supernatant from pY654–β-catenin IP was subsequently immunoprecipitated with total β-catenin antibody. Lane 3 shows p-Smad associated with pY654–β-catenin, and lane 5 shows the remainder of p-Smad2 on β-catenin after pY654–β-catenin depletion. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. All of the aforementioned experiments have been performed at least three times with similar results.