Bmpr2

Cells were lysed with ice-cold RIPA buffer (50 mM Tris-HCl, pH 7.5; 150 mM NaCl; 1% NP-40; 0.25% sodium deoxycholate, 1mM EDTA), supplemented with a protease inhibitor cocktail (Sigma-Aldrich). Equal amounts of extracts (30μg) were then electrophoresed on a sodium dodecyl sulfate (SDS) polyacrylamide gel and electroblotted to nitrocellulose filter membranes (Millipore). Then, membranes were immersed in blocking buffer (5% degreased milk powder) for 1 h and incubated with antibodies against HIF-1α, HIF-2α (Novus Biologicals, Littleton, CO), Smad5, TATA-binding protein (TBP), α-tubulin, Bmpr2, Bmpr1a (ProteinTech Group, Chicago, IL), β-actin or Smad4 (Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4°C. They were then incubated with horseradish peroxidase-conjugated secondary antibodies (Jackson Immuno-Research, West Grove, PA) and the protein bands were visualized using the SuperSignal chemiluminescent detection module (Pierce).

The proteins were isolated from pulmonary arteries or HPASMCs using radio-immunoprecipitation assay buffer (containing 0.1% phenylmethylsulfonyl fluoride), and equal proteins (30 μg) were separated by electrophoresis and moved to polyvinylidene fluoride membranes. Then the membranes were blocked with 1% BSA for 1 h, followed by overnight incubation with primary antibodies against cGKI (CST, United States), BMPR2 (Proteintech, United States), p-Smad1/5/8 (CST, United States), Id1 (Proteintech, United States), PCNA (CST, United States), and β-actin (Proteintech, United States) at 4°C. After that, the membranes were probed with HRP-labeled secondary antibodies (Jackson, United States). The signals of bands were measured using Luminata Creseendo Western HRP Substrate (Millipore) through Molecular Imager ChemiDoc XRS System (Bio-Rad, United States). The protein levels were quantified using Image J 1.43.