Figure 2

ALK1 and Smad are critically important in Smad1/5 phosphorylation in ECs. (A) BAECs were adenovirally infected with wild-type ALK1/HA or LacZ construct at MOI of 1000. Fresh medium containing 10% FBS was added 16 h after infection. Eight hours later, the cells were starved overnight and then stimulated with TGF-β (1 ng/ml) for the indicated time periods and TGF-β-induced Smad1/5 phosphorylation was measured. Cells were lysed, sonicated and fractionated by SDS-PAGE. The gels were then subjected to immunoblotting. The filters were incubated with PS1, PS2, HA or actin antibody. (B) MEECs were transfected with BRE-luc in the absence or presence of ALK1 or Smad5, or both. After 48 h, cells were extensively washed. Then, cells were stimulated for 8 h with TGF-β, or not, and luciferase activity was measured. Values are corrected for transfection efficiency as measured by β-galactosidase activity. A representative experiment using triplicate samples is shown. (C) MEECs were transfected with (BRE)-luc in the absence or presence of ALK1-RNAi. After 48 h, cells were extensively washed. Then, cells were stimulated for 8 h with TGF-β, or not, and luciferase activity was measured. The luciferase activity upon RNAi-mediated knockdown of endogenous mouse ALK1 was rescued by co-transfecting a human ALK1 expression plasmid. Values are corrected for transfection efficiency as measured by β-galactosidase activity. A representative experiment using triplicate samples is shown. (D) MEECs were transfected with BRE-luc in the absence or presence of Smad5-RNAi. After 48 h, cells were extensively washed. Then, cells were stimulated for 16 h with TGF-β, or not, and luciferase activity was measured. The luciferase activity upon RNAi-mediated knockdown of endogenous mouse Smad5 was rescued by co-transfecting a mouse Smad5 plasmid. Values are corrected for transfection efficiency as measured by β-galactosidase activity. A representative experiment using triplicate samples is shown.