Western Blot Quantification of SIRT1 and NF-κB

The Western blot procedure was described in detail by Mikłosz et al. [56 (link)] Briefly, tissue homogenate containing 30 µg of total protein was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes (0.75 A for 1 h). Then, membranes were blocked in Tris Buffer Saline Tween 20 (TBST; 20 mM Tris, 150 mM NaCl, 0.1% Tween 20) containing 5% non-fat dry milk (90 min at room temperature). Membranes were incubated overnight with primary antibodies: SIRT1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-nuclear factor-κB (NF-κB) (Cell Signaling Technology, Leiden, The Netherlands). Next, SIRT1 and NFκB were detected with antirabbit IgG horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology). The protein bands were visualized using a chemiluminescence substrate (Thermo Scientific, Waltham, MA, USA) and quantified by densitometry (Bio-Rad Systems). The protein expression was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Santa Cruz Biotechnology) expression.