Fig 1. BMP9 response and western blot analysis of ALK1 novel mutants.

(A) Schematic localization of the 14 novel mutations on the ALK1 protein. The exons in which the nucleic acid substitutions occur and their resulting amino acid substitutions are indicated. The mutations are distributed in the extracellular and the kinase domain of the ALK1 receptor. (B) Functional analysis of the ALK1 protein variants was performed by measuring luciferase reporter activity after BMP9 stimulation, as explained in materials and methods section. 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. (C) Western blot analysis of ALK1 protein variants was performed after transient transfection of HeLa cells by expression vectors encoding a C-terminally HA-tagged version of either WT-ALK1 or the different novel ALK1 mutants. Transfection with an empty pcDNA3 vector was used as control. After 48 hours, cells were lysed and 50 μg of cell lysates were resolved by 10% SDS-PAGE and immunoblotted with antibodies against HA or against βactin as a loading control.