Ab11734

The protein expression of ACE and ACE2 in the soleus and plantaris muscles was analyzed using western blot. The frozen samples were homogenized in cell lyses buffer containing 100 mM Tris-HCl, 50 mM NaCl, 1% Triton X-100, and protease inhibitor cocktail (1∶100, Sigma-Aldrich, USA). After centrifugation (10,000 × g, 4°C, 10 min), the pellet was discarded, and the samples were loaded (Laemmli 1∶1, Sigma-Aldrich, USA) and underwent SDS-PAGE in 10% polyacrylamide gels. Equal loading of samples (30 µg) were applied for electrophoresis, and proteins were electro-transferred to nitrocellulose membrane (BioRad Biosciences, USA). The blot membrane was then incubated in a blocking buffer (5% BSA, 10 mM Tris-HCl, pH 7.6, 150 mM NaCl, and 0.1% Tween 20) for 2 hours at room temperature and then incubated overnight at 4°C with mouse anti-ACE (ab11734, 1∶100, Abcam, USA) and rabbit anti-ACE2 (sc-20998 1∶200, Santa Cruz, USA). Binding of the primary antibody was detected with the use of peroxidase-conjugated secondary antibodies, and enhanced chemiluminescence reagents (Amersham Biosciences, USA) were used to visualize the autoradiography. Quantification blot analyses were performed using Image-J software (National Institute of Health, USA), normalized to relative changes in mouse anti-GAPDH (ab9484, 1∶5000, Abcam, USA).

The livers and arteries were homogenized with lysis buffer. The homogenized tissues in the lysis buffer were sonicated (NRE-02; Next Advance, Troy, NY, USA), centrifuged at 18,033 × g 4°C for 20 min, and the supernatant was used for biochemistry assays. The concentration of proteins was quantitated by the Bradford protein assay. The samples were separated using 10% SDS-PAGE, transferred to polyvinylidene fluoride membranes, and blocked with 5% skim milk dissolved in Tris-buffered saline containing 0.1% Tween 20 for 1 hr at room temperature. Then, the membrane was incubated with antiangiotensinogen (ab213705, dilution 1 : 1000; Abcam plc, Cambridge, UK), anti-ACE I (ab11734, dilution 1 : 1000; Abcam plc), anti-eNOS (ab76198, dilution 1 : 1000; Abcam plc), and anti-β-actin (ab20272, dilution 1 : 1000; Abcam plc) primary antibodies overnight at 4°C, followed by horseradish peroxidase conjugated goat anti-mouse (ab205719, dilution 1 : 1000; Abcam plc) and goat anti-rabbit (ab205718, dilution 1 : 1000; Abcam plc) secondary antibodies were treated and reacted at room temperature for 1 hr. After the reaction, the membrane was visualized with ECL solution (EzWestLumi plus; ATTO, Amherst, NY, USA) which reacts with horseradish peroxidase and was analyzed using Image J software (version 1.49; Bethesda, MD, USA).