Expression and localization of the different ALK1 mutants. (A) The 19 different mutations are distributed on the map of the ALK1 protein. The mutations indicated with a dotted line are the conflicting mutations. (B) Protein expression of ALK1 mutants. Expression vectors encoding HA-tagged (at their C-terminus), ALK1 (WT or mutants), or β-gal (control) were transiently transfected into NIH-3T3 cells for 24 hours. Cell lysates (20 μg proteins) were resolved by 10% SDS-PAGE and immunoblotted with antibodies directed against HA and α-tubulin (as a loading control). (C-D) Flow cytometry analysis of NIH-3T3 cells transfected with expression vectors encoding HA-tagged (at their N-terminus), ALK1 (WT or mutants), or β-gal (control) for 24 hours. Nonpermeabilized transfected cells were stained for cell surface ALK1 expression (C, anti-ALK1 antibody) or (D, anti-HA antibody).