Western blotting of ACE, ACE2 and the MAS receptor

Tissue (100 mg) was 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]) and centrifuged at 15,000 × g for 20 min at 4°C. The protein (100 µg) was subjected to SDS-PAGE with a 10% polyacrylamide gel and transferred onto a PVDF membrane (Pall, Port Washington, NY, USA). The 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-ACE, anti-ACE2 (1:500 dilution; Millipore, Billerica, MA, USA), or anti-MAS (1:1,000 dilution; Millipore) antibodies, and then incubated with horseradish peroxidase-conjugated secondary antibody. Signals were detected using the Western Lighting® chemiluminescent kit (Millipore) according to the manufacturer's specifications.