Enhanced chemiluminescent kit

WB was carried out as previously described [8 (link), 9 (link), 43 (link)]. Membranes containing transferred proteins were incubated with primary antibodies (1:1000) in 5% (wt/vol) Blotto and subsequently with the appropriate horseradish peroxidase-conjugated secondary antibody (1:2000) in 5% (wt/vol) Blotto (Supplementary Table 4 for details on antibodies). Peroxidase activity was visualized on a film with the Enhanced Chemiluminescent Kit (Amersham Biosciences) and signal intensity densitometrically determined (Image J software).

Protein expression in total lung tissues, HPMECs, and PASMCs were determined by Western blotting. After transferation, the PVDF membranes (Millipore, United States) were blocked with 5% skim milk to prevent non-specific binding, then incubated with specific primary antibodies against HIF1-α (1:500, ab216842, Abcam, United States), NOX4 (1:1,000, 14347-1-AP, Proteintech, China), SOD2 (1:1,000, 24127-1-AP, Proteintech, China), SOD1(1:1,000, 10269-1-AP, Proteintech, China), Cleaved Caspase-3 (1:1,000, 9664, Cell Signaling Technology, United States), Bcl-xL (1:1,000, AB126, Beyotime, China), β-actin (1:2,500, 60008-1-Ig, Proteintech, China). After washing with TBST buffer, the membranes were incubated with appropriate secondary antibody conjugated with horseradish peroxidase, and signals were detected using by enhanced chemiluminescent kit (Amersham Biosciences, United Kingdom). The relative OD of Western blotting was calculated with the Image lab software (Bio-Rad Laboratories, Hercules, CA, United States).