BMP9 binding to the different ALK1 mutants and functional analysis of the NANDOR ALK1 mutants. (A) β-gal, WT-ALK1, or the different ALK1 mutants were transiently transfected in COS-7 cells for 48 hours. Cells were then incubated with [¹²⁵I]BMP9 for 2 hours at 4°C. Cells were then washed, lysed, and the bound radioactivity was counted as described in “[¹²⁵I]-BMP9 radioreceptor assay.” Data are mean ± SD from 1 representative experiment of 3. (B) NIH-3T3 cells were transiently transfected with pGL3(BRE)₂-luc, pRL-TK-luc, and WT-ALK1, or the constitutively active ALK1 mutant (ALK1ca, Q201D) or the different ALK1 double mutants (0.5 ng) or β-gal (control). After 4 hours, cells were treated or not with BMP9 (100 pg/mL) for 15 hours. The luciferase activities were then measured as described in “DNA transfection and dual luciferase activity assay.” The relative firefly luciferase activity was normalized to renilla luciferase activity. Data are mean ± SD of 3 independent experiments. (C) COS-7 cells were cotransfected with expression vectors encoding an HA-tagged and a His-tagged version of either WT-ALK1 or ALK1 mutants for 24 hours. Cell lysates (1-mg proteins) were subjected to immunoprecipitation with anti-His. These lysates were then resolved by 10% SDS-PAGE and immunoblotted with antibodies against HA (IP/IB-HA). In parallel, cell lysates (20 μg proteins) were also resolved by 10% SDS-PAGE and immunoblotted with antibodies against HA (IB-HA).