Ab216476

Kidney sample was homogenized in RIPA buffer by smashing in liquid nitrogen. Same amount of lysates protein (40 µg) was loaded and separated in SDS-PAGE. The resolved proteins were transferred onto nitrocellulose membranes and probed with specific primary antibody at 4°C overnight with agitation. GAPDH was probed as a loading control. The protein bands were visualized using enhanced chemiluminescence (ECL) reagents (GE Healthcare, Chicago, Illinois, United State) and developed in Fujitsu Biomedical film (Fujitsu, Japan). Densitometric analysis was performed using the ImageJ (NIH). Quantification protein expression was based on the ratio of target protein to GAPDH. The following primary antibodies were used: anti-ACE (Ab216476, Abcam, 1:500), anti-FGF2 (05-118, Millipore, 1:1000), anti-NHE3 (NB110-61586, Novus Biological, 1:2000), anti-Rac1 (Ab33186, Abcam, 1:1000), anti-Nr3c2 (Ab2774, Abcam, 1:1000, anti-Sgk1 (Ab59337, Abcam, 1:1000), anti-β-tubulin I (T7816, Sigma-Aldrich, 1:30000), and anti-GAPDH (2251-1, Epitomics, 1:30000).

Fifty micrograms of cell lysates and myocardial tissue were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and then transferred onto nitrocellulose membranes. Anti-Angiotensin converting enzyme 1 antibody (ACE) (ab216476) and anti-Angiotensin II type 1 receptor antibody (AT1R) (ab18801) were purchased from Abcam, Cambridge, UK. HRP conjugated immunoglobulin was used as a secondary antibody (Jackson ImmunoResearch Laboratories). West Pico Chemiluminescent (Pierce) was used as the substrate to visualize protein bands and quantified using densitometry image analysis software (Image Master VDS; Pharmacia Biotech).