BMP9 activates the Smad1/5/8 pathway in HMVEC-d's. (A-B) HMVEC-d's were treated with BMP9 for different times and at different concentrations. Cell lysates (20 μg proteins) were resolved on a 10% SDS–polyacrylamide gel, and immunoblotted with antibodies against phospho-Smad1/5/8 or against α-tubulin. (C-D) HMVEC-d's were transiently transfected with pGL3(BRE)-luc and pRL-TK-luc. After 4 hours, cells were treated with different concentrations of BMP9 (0.01, 0.1, 1, or 10 ng/mL) in panel C and BMP9 (1 ng/mL) or TGFβ1 (1 ng/mL) in panel D for 15 hours. (E) HMVEC-d cells were transiently transfected with hALK1 siRNAs or control (CTL) siRNA together with pGL3(BRE)-luc and pRL-TK-luc. After 4 hours, cells were treated with BMP9 (1 ng/mL) for 15 hours. The luciferase activities were then measured as described in “Materials and methods.” The relative firefly luciferase activity was normalized to renilla luciferase activity. Data shown in panels C-E are representative of 1 experiment ± SD of 3. Inset in panel E represents ALK1 protein level revealed by Western blotting with an anti-ALK1 antibody after immunoprecipitation with another antibody directed against ALK1 of 500 μg total protein obtained from cells treated 48 hours with control siRNA or ALK1 siRNA. (F) H5V cells were transiently transfected with mALK1 siRNAs or CTL siRNA together with pGL3(BRE)-luc or pRL-TK-luc, with or without human pALK1. After 4 hours, cells were treated with BMP9 (0.1 ng/mL) for 15 hours. The firefly luciferase activity was normalized to renilla luciferase activity. Results are the mean ± SE of 3 experiments; *P < .05.