Anti ace

Total protein in the adipose tissue of 6- and 16-week-old rat offspring was extracted, and the protein concentration was measured by the bicinchoninic acid (BCA) method. After denaturation and electrophoresis on sodium dodecyl sulfate (SDS)-polyacrylamide gels, separated proteins were transferred to nitrocellulose membranes. Membranes were then blocked in 5% nonfat milk in TBST for 1 h. After incubation with primary antibodies [anti-AT1-R (Abcam, UD), anti-AT2-R (Abcam, UK), anti-ACE (Santa Cruz, USA) or anti-β-actin (Sigma, USA)] in TBS at 4°C overnight, membranes were incubated with peroxidase-conjugated secondary antibody in TBS at room temperature for 1 h. Specific bands were detected with a chemiluminescence assay and recorded on X-ray film. The Quantity One software (Bio-Rad, USA) was used to quantify the band intensities.

Protein expression levels of ACE, ACE2, AT1R, and MasR in cardiac tissue were determined using western blotting as previously described [24 (link)]. In brief, frozen cardiac tissues were thawed, ground, and homogenized in the lysis buffer that contained protease inhibitors, surfactants, as well as phosphatase inhibitors. Protein samples were loaded in 8% or 10% gels for sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The resolved proteins were electrically transferred to the nitrocellulose membranes and detected using specific antibodies, including anti-ACE (SantaCruz Biotechnology, Inc., CA, USA; cat no. sc-23,908; lot no. C1319), anti-ACE2 (Abcam, UK; cat no. ab108252; lot no. GR145000–28), anti-AT1R (SantaCruz Biotechnology, Inc., CA, USA; cat no. sc515884; lot no. J0319), and anti-MasR (SantaCruz Biotechnology, Inc., CA, USA; cat no. sc-390,453; lot no. A1419), to quantify myocardial protein levels. The primary antibody-antigen complexes and the horseradish peroxidase (HRP)-conjugated secondary antibodies were incubated and then visualized using an enhanced chemiluminescence (ECL) substrate (Thermo Fisher Scientific Inc., USA) and ECL hyperfilm (GE Healthcare Pvt. Ltd., UK). Blots were inspected by ImageJ software (NIH, USA). Results were normalized with the beta-actin (Sigma-Aldrich, USA; cat no. A5441) as the internal standard.

DMEM-F12, newborn bovine serum, HEPES and penicillin/streptomycin were purchased from Wisent Corporation (Wisent, Canada). Hank's balanced salt solution (HBSS), nonessential amino acids, sodium pyruvate, insulin-transferrin-selenium and L-glutamine were purchased from Invitrogen Life Technologies (Paisley, Scotland). Stainless steel sieves were obtained from Merck Eurolab (Leuven, Belgium). Anti-NCC (Stressmarq Biosciences Inc., Canada), anti-NHE3 (Abcam, Cambridge, MA), anti-AGT (Abcam, Cambridge, MA), anti-ACE (Santa Cruz Biotechnology, Santa Cruz, CA), and anti-MnSOD (Santa Cruz Biotechnology, Santa Cruz, CA) primary antibodies and horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) were used.