Figure 1

Negative regulation of TGF-β-induced Smad1/5 phosphorylation.(A) First panel. BAECs were stimulated with TGF-β (1 ng/ml) for different time periods at 37°C before lysis. Whole cell lysate was sonicated and fractionated by SDS-PAGE and blotted. The filters were incubated with PS1 antibody that specifically recognizes phosphorylated Smad1/5, PS2 antibody, which specifically recognizes phosphorylated Smad2, and antisera against Smad5. The blots were incubated with an actin antibody as a control for protein loading. Second panel. BAECs were treated with the protein synthesis inhibitor cyclohexamide (10 ng/ml) 30 minutes before they were stimulated with TGF-β. The filters were incubated with PS1, PS2 or actin antibodies. Third panel. BAECs were treated with the proteasome inhibitor, MG-132 (10 nM), for 30 min before they were stimulated with TGF-β. The filters were incubated with PS1, PS2 or actin antibodies. Last panel. BAECs were pre-treated with a phosphatase inhibitor, sodium orthovanadate (1 mM) 30 min prior to stimulation with TGF-β following the same procedure as in Fig. 1A. The filters were incubated with PS1, PS2 or actin antibody. (B) BAECs were pre-treated (right panel) or not (left panel) with the Ser/Thr phosphatase inhibitor calyculin (1 nM) 30 min prior to stimulation with TGF-β for different time periods before lysis. The filters were incubated with PS1 or actin antibody. (C) MEECs were pre-treated with cyclohexamide, MG-132, vanadate or not, and then stimulated with TGF-β for different time periods as in Fig. 1A.