Sc514211

Whole cell lysates and western blotting were performed as described previously 9 (link). Antibodies against CD105 (ab169545, 1:1000), Smad2 (ab40855, 1:2000), pSmad3 (ab52903, 1:1000) were purchased from Abcam. An antibody against BMP9 (sc514211, 1:500) was purchased from Santa Cruz Biotechnology (USA). Antibodies against Smad1 (6944, 1:1000), pSmad1/5/8 (13820, 1:1000), and pSmad2 (3108, 1:1000) were purchased from Cell Signaling Technology. Antibodies against Smad3 (YM3417, 1:2000), GAPDH (YM3029, 1:20000), and β-tubulin (YM3030, 1:5000) were purchased from ImmunoWay Biotechnology.

Cell lysates were thawed, sonicated at 10 µm amplitude for 10 s, and then cleared by centrifugation. A modified Lowry assay was carried out (BioRad) to quantify the protein concentration of each sample. Conditioned media (60 µL) or lysates (30 µg of total protein) were mixed with 5x SDS loading buffer (312.5 mM Tris–HCl, pH 6.8, 10% SDS, 50% glycerol, 0.0075% bromophenol blue, and 12.5% β‐mercaptoethanol) and boiled at 98°C for 5 min to denature and reduce proteins. Samples were then analyzed by gel electrophoresis and western blotting. For the growth factor domain, samples were blocked in 5% BSA and probed using sc‐514211 (1:1000; Santa Cruz Biotechnology). For the prodomain, samples were blocked in 5% non‐fat milk protein (Marvel) and probed using AF3879 (1:2500; R&D Systems). Lysate blots were re‐probed using an antibody toward β‐actin (A5441, clone AC‐15; Sigma Aldrich).