Figure 3

Smad7 is a potent inhibitor of ALK1-induced Smad1/5 phosphorylation in Ecs.(A) MEECs were stimulated with TGF-β or adenovirally infected at MOI of 500 with LacZ, caALK1, caALK5 and Smad7 (included as a positive control). Forty hours later, the noninfected cells were starved overnight and then stimulated with TGF-β, or not, for 90 min. Cells were lysed, and RNA was isolated and cDNA prepared. Expression of Smad7 was analysed by semi-quantitative RT-PCR. β-actin expression was measured to control for equal loading. The PCR products were loaded on 1% agarose gel and stained with ethidium bromide. (B) Left panel. BAECs were infected with adenoviral constructs of Flag-Smad7 or lacZ as a control with MOI of 1000. Forty hours later, BAECs were stimulated with a different dose of TGF-β at 37°C before lysis. Whole cell lysate was sonicated and fractionated by SDS-PAGE and blotted. The filters were incubated with PS1, PS2 or α-Flag antibody. Right panel. Differential Smad7 inhibition on TGF-β induced Smad1/5 phosphoylation and Smad2 phosphorylation in BAECs. BAECs were infected with adenoviral constructs of Flag-Smad7, or LacZ as a control, with MOI of 400. Forty hours after infection, BAECs were stimulated with 1 ng/ml of TGF-β 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, PS2 and Flag antibodies. (C) MEECs were co-transfected with caALK1 and BRE-luc with or without Smad7 at different concentrations. Luciferase activity was measured 48 h after transfection. Values are corrected for transfection efficiency as measured by β-galactosidase activity. A representative experiment using triplicate samples is shown. (D) Smad7-RNAi plasmid with a hygromycin cassette was stably transfected in MEECs and selected on hygromycin medium for 7 days. PGK-hygromycin selected cells were used as mock cells. The cells were serum-starved overnight and stimulated with TGF-β (1 ng/ml) at different time periods prior to lysis, sonication and fractionation by SDS-PAGE. Gels were then subjected to immunoblotting. The filters were incubated with PS1, PS2, or actin antibody. (E) Left panel. MEECs were transfected with BRE-luc in the absence or presence of Smad7-RNAi. Forty-eight hours after transfection the cells were washed extensively. Luciferase activity was measured after 8 hours of stimulation of TGF-β or not. Values are corrected for transfection efficiency as measured by β-galactosidase activity. A representative experiment using triplicate samples is shown. Right panel. MEECs were transfected with (CAGA)₁₂-luc in the absence or presence of Smad7-RNAi. Forty-eight hours after transfection the cells were stimulated for 16 hours with TGF-β and luciferase activity measured. A representative experiment using triplicate samples, corrected for transfection efficiency, is shown.