Fig 2: ALK-1 colocalizes, interacts, and co-sediments with CAV-1.A, representative confocal images of CAV-1 (red channel) and ALK-1 (green channel) in HUVEC; DAPI was used to visualize the nucleus. Highlighted areas reflect magnified areas from four different cells in the upper panel. Scale bars = 10 μm. B, HUVEC were treated with BMP-9 (0.5 ng/ml) for 30 min, ALK-1 immunoprecipitated using antibody against ALK-1 or isotope-matched control IgG. C, membrane separation on a sucrose gradient prepared from HUVEC depicting light membranes floating in fractions 3–5 and heavy membranes in fractions 8–12.

Fig 3: BMP-9 triggers ALK-1 internalization.A, HUVEC were treated with a range of human BMP-9 for 30 min, and biotin-labeled proteins on the plasma membrane were isolated and immunoblotted for ALK-1 or VECAD. B, quantitative analysis of ALK-1 on the cell surface normalized to VECAD levels. C, HUVECs were treated with BMP-9 (10 ng/ml) for indicated times, and cell surface biotin-labeled protein analyzed. D, quantitative analysis of results of ALK-1 on the cell surface normalized to VECAD levels. Data represented three or four independent experiments, and quantification represents the mean ± S.E. Statistical significance was assessed by one-way ANOVA (*, P ≤ 0.05 when significant).

Fig 4: LDL suppresses BMP-9/ALK-1 signaling.A, HUVECs were incubated with different amounts of LDL for 16 h, then treated with BMP-9 (0.5 ng/ml) for 30 min. B, quantification p-SMAD/SMAD1 from three independent experiments. C and D, HUVECs were transfected with Scr or ALK-1 siRNAs then cultured in LPDS or LPDS containing LDL (25 µg/ml) for an additional 16 h. Treated cells were then incubated with differing amounts of BMP-9 (0.01-10 ng/ml) to assess p-SMAD1/5 phosphorylation and quantified in D. E and F, kinetics of SMAD1/5 phosphorylation in the absence and presence of LDL. HUVECs were incubated in LPDS or in LPDS plus LDL (25 μg/ml) for 16 h and then stimulated with BMP-9 (0.5 ng/ml) for indicated time and quantified in F. G, SMAD1/5-dependent gene expression is reduced in LDL pretreated cells. HUVECs were treated with LDL (25 μg/ml for 16 h) and then incubated with BMP-9 (0.5 ng/ml) for 8 h and qPCR analysis performed. All quantitative data were from three independent experiments.

Fig 5: LDL triggers the endocytosis of ALK-1.A, HUVECs were incubated with LDL (25 μg/ml) for the indicated times and biotinylated cell surface proteins isolated for immunoblotting. B–E, levels of cell surface LDLR, ALK-1, and SR-B1 after treatments of HUVEC with LDL (B, 25 μg/ml) or BMP-9 (D, 0.5 ng/ml) for the indicated time (0–30 min) and quantified in C and E. F and G, HUVEC were transfected with siRNAs for 48 and incubated with LDL (25 μg/ml) for 60 min; ALK-1 on the cell surface detected using biotin-labeling and the quantified G. Quantitative data were from three independent experiments.