Fig. 5. BMP-9 activates the Smad1- and Smad5-responsive BRE luciferase reporter through ALK1. (A-B) BAECs were transiently transfected with an ID1-promoter-derived luciferase reporter construct (BRE) and a construct for -galactosidase as internal expression control. Before stimulation, cells were serum-starved for 8 hours and then incubated with the respective ligands overnight. (A) BRE activity is induced by BMP-9 (100 ng/ml) and BMP-6 (100 ng/ml) but not by TGF- (5 ng/ml). (B) BMP-9 (5 ng/ml) stimulated BRE-induction can be inhibited by a 15-fold molar excess of ALK1-Fc. (C,D) shRNA constructs against ALK1 or ALK2 were cloned into the pSuper vector and co-transfected into BAECs. ALK1 shRNA blocks BMP-9-stimulated (5 ng/ml) (C), but not BMP-6-induced (25 ng/ml) BRE activation (D). (E) ALK1 shRNA-mediated reduction of BRE activity is rescued by overexpression of human ALK1, but not by human ALK2. ^*P<0.01 compared with BMP-9-stimulated control; ^#P<0.01 compared with ALK1 RNAi after BMP-9 stimulation.