Effect of HHT2-related ALK1 mutations on the cell surface distribution of the receptor and its ability to activate Smad1. (A) HEK293T cells transfected with plasmids encoding HA-tagged wild-type or mutant ALK1 and His-tagged TβRII were treated with 200 pM TGFβ1 for 30 minutes. Protein complexes were isolated from the cell lysates by precipitation with Ni-NTA beads and detected by immunoblotting (IB) with anti-HA antibodies (top panel). Protein expression was confirmed by immunoblotting with anti-HA and anti-His antibodies (middle and bottom panels). (B) HEK293T cells transfected with plasmids encoding HA-tagged wild-type or mutant ALK1 and Flag-tagged Smad1 lacking the C-terminal 8 amino acids (shown to associate with active receptor⁷ ), were treated with 200 pM TGFβ1 for 30 minutes. Protein complexes were isolated from the cell lysates by immunoprecipitation (IP) with an anti-Flag antibody and detected by immunoblotting with an anti-HA antibody (top panel). Protein expression was confirmed by immunoblotting with anti-HA and anti-Flag antibodies (middle and bottom panels). (C) Cells transfected with HA-tagged wild-type or mutant ALK1 were biotinylated with 0.5 mg/mL NHS-SS-biotin for 40 minutes at 4°C. Biotinylated surface receptors were precipitated with ImmunoPure Streptavidin beads and analyzed by anti-HA immunoblotting. (D) COS7 cells transfected with the plasmids encoding HA-tagged ALK1 wild-type, ALK1 mutants, Flag-tagged Smad1, and His-tagged TβRII were treated with 200 pM TGFβ1 for 30 minutes. The cells were subsequently lysed and the phosphorylated Smad1 was detected by immunoblotting (IB) with anti–phospho-Smad1 antibodies (upper panel). Total protein expression was confirmed by immunoblotting with anti-Smad1 and anti-HA antibodies (middle and bottom panels). The active form of BMP type I receptor BMPRIB(QD) was used as a positive control for induction of Smad1 phosphorylation.