ALK1 signals independently of ALK5 signaling. (A) BAECs were transfected with the BRE-lux luciferase reporter gene, and treated with combinations of BMP-9 (10 ng/mL), TGFβ1 (1 ng/mL), the ALK5 inhibitor SB-431542 (5 μM), and the BMP-9 inhibitor ALK1/Fc (200 ng/mL). After 24 hours, ALK1 signaling was determined by luciferase activity. Asterisks indicate statistically significant differences compared with control (no treatment). ***P < .001; Tukey test. (B) BAECs were treated with the same treatments as in panel A, and samples were collected after the indicated times. Activation of SMAD1/5/8 was determined by pSMAD1/5/8 immunoblotting and compared with total SMAD. (C) HAECs were transfected with scrambled (SCR) siRNA, or siRNA to ALK1 or ALK5. The following day, the cells were left untreated or treated with BMP-9 (10 mg/mL) or TGFβ1 (1 ng/mL) for 1 hour. Activation of SMAD1/5/8 was determined by pSMAD1/5/8 immunoblotting and compared with total SMAD.