TGF-β1 promotes the serine phosphorylation of endoglin in ECs. (A) HAECs were serum-starved for 2 hours, then pretreated with (+) or without thrombin (3 U/mL) for 30 minutes followed by stimulation with TGF-β1 (2 ng/mL) for various time periods as indicated. Lysates were immunoblotted with the antibody against phospho–NF-κB p65 (Ser-536) that also detects the serine-phosphorylated endoglin as described in Figure 1. The serine phosphorylation of endoglin by TGF-β1 was shown as fold increase relative to control cells by densitometric analysis. The same blot was stripped and reprobed with an endoglin antibody (SN6h) to show the equal loading of lysates (lower panel). (B) HAECs were left untreated (control) or pretreated with SB431542 (10 μM) for 30 minutes, then stimulated with TGF-β1 (2 ng/mL) for 5 minutes. Lysates were subjected to immunoblotting with indicated antibodies against the serine-phosphorylated endoglin as described in Figure 1, endoglin (SN6h), phospho-Smad2 (Ser-465/467), or Smad2. Results shown are representative immunoblots of 3 independent experiments.