Western blot analysis of SIRT1 protein expression

Myocardial tissue samples belonging to the same group (first group: CABG n=6 vs. control n=6; second group MACCE (−) CABG n=6 vs. MACCE (+) CABG n=6) were used for Western blot analysis. The CABG patients in the first group were selected from patients without any MACCEs. The control group included patients with all types of valve disease.

Each sample was homogenized separately. After treatment with lysis buffer (RIPA Lysis Buffer, Thermo Fisher Scientific, Cat. No. 89900) and protease inhibitor and phosphatase inhibitor cocktail, the protein concentration was measured with the Quibit^® Fluorimeter (Invitrogen, Life Technologies Corporation, Carlsbad, CA, USA). Forty μg protein was size-fractionated by 4–12% NuPAGE electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes using the iBlot^® Dry Blotting System (Invitrogen, Life Technologies Corporation). After blocking the membrane in 5% nonfat milk in 50 mm Tris-buffered saline for 1 h at room temperature, it was incubated overnight with rabbit polyclonal anti-sirt1 (rabbit polyclonal, 07-131, Millipore). Thereafter a peroxidase-conjugated goat anti-rabbit (Amersham, GE Health Care UK Limited, Buckinghamshire, England) antibody was used as the second antibody. To test the reproducibility, Western blot analysis was performed for each sample 3 times.

Protein loading was controlled using the rabbit monoclonal GAPDH antibody (rabbit monoclonal antibody, 14C10, Cell Signaling). The ECL-Advanced Western Blotting Detection kit (Amersham, GE Health Care UK, Limited) was used for blot development and the Bio-Rad ChemiDoc XRS (Bio-Rad Laboratories, Inc) was used for visualization. Protein quantification was conducted densitometrically with the ImageJ program and corrected with values determined on GAPDH blots. The expression was given as relative values compared with the control or MACCE negative-CABG groups. The t test was used for statistical analysis. The results are shown as mean ± standard deviation (SD). * p<0.05. ** p<0.001.