BMP10 is also an activator of ALK1. (A) BMP10 with an N-terminal His₆ tag was purified from conditioned medium of 293T cells using metal chelate chromatography. BMP10 was electrophoresed on polyacrylamide gels under reducing conditions and detected by immunoblotting using antibodies against polyhistidine (His₆). (B-C) NIH-3T3 cells were transiently transfected with pGL3(BRE)-luc, pRL-TK-luc, and either pCDNA₃ empty vector or pALK1. After 4 hours, cells were treated with BMP9 (0.1 ng/mL) or BMP10 (10 ng/mL) in the presence or absence of ALK1ecd (15-fold molar excess over BMP10 concentrations) for 15 hours. The firefly luciferase activity was normalized to renilla luciferase activity. Results are presented as mean values ± SD. (D) HMVEC-d monolayers were scratched to create a wound and incubated under low serum concentration (0.5% FBS) in the presence or absence of BMP10 (100 ng/mL). At time 0, 24, and 48 hours after wounding, the cells were observed by phase-contrast microscopy and photographed. Results from 1 representative experiment of 3 are presented as the percent of wound closure at different times after wounding ± SE. (E) HMVEC-d's were treated for 24 and 48 hours with BMP10 (100 ng/mL) in 5% FBS. The quantity of viable cells was determined using the WST-1 assay. Results from 1 representative experiment of 3 are expressed as absorbance (OD₄₅₀) ± SE. (D-E) **P < .01.