Thrombin down-regulates TGF-β signaling in ECs. (A-C) HUVECs were left untreated (control) or pretreated with thrombin (3 U/mL), TRAP (40 μM), or Ang II (100 nM) for 30 minutes or as indicated or preincubated with thrombin (3 U/mL) for 16 or 24 hours, then stimulated with TGF-β1 (2 ng/mL) for 25 minutes. Lysates were subjected to immunoblotting with antibodies against phospho-Smad2 (Ser-465/467), Smad2, ALK5, TβRII, or endoglin. The serine phosphorylation of Smad2 by TGF-β1 was shown as the percentage relative to control cells stimulated with TGF-β1 alone by densitometric analysis from 3 independent experiments. (D) HUVECs grown on glass coverslips were left untreated (control) or pretreated 30 minutes with thrombin (3 U/mL) or TRAP (40 μM), then stimulated with TGF-β1 (2 ng/mL) for 20 minutes. Cells were fixed and stained with a monoclonal antibody to Smad2/3 (10 × 100). Results shown are representative of 3 independent experiments.