Ab213705

Western blot was performed as previously described [17 (link)] using primary antibodies including active-β-catenin (Millipore, 05-665), AGT (Abcam, ab213705), ACE-1 (Abcam, ab222739), AT1 (Abcam, ab18801), FN (Abcam, ab2413), collagen-I (Boster, BA0325), and α-SMA (Sigma, A5228).

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).