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.