Chemidoc image acquisition and analysis tool

For total protein extraction, cells were directly lysed in a buffer containing 50 mM Tris/HCl at pH 7.5, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 25 mM NaF, 25 mM β-glycerolphosphate, 0.1 mM Na₃VO₄, 0.1 mM PMSF, 0.2% Triton X-100, 0.3% Nonidet P40, and a cocktail of protease inhibitors (100 X, EuroClone). After incubation on ice for 30 min, the lysate was centrifuged at 13,000 rpm for 15 min at 4°C. A total of 15 μg of protein per well was separated by 4–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Membranes were then blocked with 5% milk-TBST buffer (TBS plus 0.1% Tween-20) for 1 h at room temperature and incubated with primary antibodies overnight at 4°C, washed with TBST buffer three times, and incubated with corresponding secondary antibodies at room temperature for 45 min. Signals were detected by the “Pierce ECL Western Blotting Substrate” method (Thermo Fisher Scientific) and analyzed by the ImageLab software, using the Chemidoc image acquisition and analysis tool (BioRad).

Total proteins were extracted from the aortic and heart tissues using a RIPA buffer with protease inhibitors, and quantified using the Bradford protein assay.
A total of 45 μg protein per well was separated via electrophoresis on 4–15% sodium dodecyl sulfate polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were then blocked with 5% milk TBST buffer (TBS plus 0.1% Tween-20) for 1 h at room temperature, and incubated with the primary antibody, anti-SIRT1 (D1D7) (Cell Signaling Technology, Inc., Danvers, MA, USA) or anti-GAPDH (G-9) (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) overnight at 4 °C, washed three times with the TBST buffer and incubated with the corresponding secondary antibodies at room temperature for 45 min, anti-rabbit and anti-mouse IgG heavy and light chain (Bethyl Laboratories, Inc., Montgomery, TX, USA), respectively. The signals were detected using the “Enhanced Chemiluminescent Substrate” method with ECL Star (EuroClone S.p.a, Milan, Italy) and analyzed with the ImageLab software, using the Chemidoc image acquisition and analysis tool (Bio-Rad Laboratories, Inc., Hercules, CA, USA). All data were expressed as the mean ± SD.