Functional analysis of the BMP9 response of conflicting ALK1 mutants. (A) Protein expression of WT-ALK1 or ALK1 mutants. Expression vectors encoding HA-tagged WT-ALK1 or ALK1 mutants or β-gal (control) were transiently transfected in NIH-3T3 cells for 24 hours. Cell lysates (20 μg proteins) were resolved by 10% SDS-PAGE and immunoblotted with antibodies against HA and α-tubulin (as a loading control). (B) Expression vectors encoding WT-ALK1 or the different ALK1 mutants or β-gal (control) were transiently transfected into NIH-3T3 cells. After 24 hours, cells were serum deprived for 1 hour and subsequently treated with BMP9 (100 pg/mL) for 1 hour. Cell lysates (20 μg proteins) were resolved by 10% SDS-PAGE and immunoblotted with antibodies against phosphoSmad1/5 or against α-tubulin (as a loading control). (C) NIH-3T3 cells were transiently transfected with pGL3(BRE)2-luc, pRL-TK-luc, and plasmids (0.5 ng) encoding either WT-ALK1 or the different ALK1 mutants or β-gal (control). After 4 hours, cells were treated (▬) 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. Results are expressed as fold induction over the value obtained for each ALK1 mutant in the absence of BMP9. Data are mean ± SD of 3 independent experiments.
