Western blot analysis of pulmonary eNOS, ACE, ACE2, and MAS
Lung tissues(100 mg) were homogenized in 1 ml of RIPA buffer (1% Triton X-100, 15 mM HEPES-NaOH [pH 7.5], 0.15 mM NaCl, 1% sodium deoxycholate, 0.1% SDS, 1 mM sodium orthovanadate, 10 mM EDTA, and 0.5% protease inhibitor cocktail [Sigma, St. Louis, MO, USA]) and centrifuged at 15,000 × g for 20 min at 4°C. Proteins in the supernatant (100 µg) were subjected to SDS-PAGE in 10% polyacrylamide gels and were transferred onto PVDF membranes (Pall, Port Washington, NY, USA). Each membrane was blocked with 5% non-fat dry milk in Tris-buffered saline (TBS), probed with anti-actin (1:10,000) (Upstate Biotechnology, Lake Placid, NY, USA), anti-eNOS ( 1:1000, Millipore, Milford, MA, USA) anti-ACE, anti-ACE2 (1:500 dilution; Millipore, Billerica, MA, USA), or anti-MAS antibodies (1:1,000 dilution; Millipore), and then incubated with horseradish peroxidase-conjugated secondary antibody. (Leadgene Biomedical, TW) Signals were detected using the Western Lighting® chemiluminescent kit (Millipore) according to the manufacturer’s specifications.