Western Blot Analysis of p-AMPK and SIRT1

Western blotting was performed as described in the previous studies [33 (link),34 (link),35 (link)] with minor modifications. Protein samples were separated via 10% SDS-PAGE and electroblotted onto polyvinylidene difluoride membranes (Millipore) for 3 h at 300 mA. The membranes were blocked with 5% nonfat dry milk or BSA dissolved in Tris–HCl saline buffer containing 0.1% Tween-20 (TBS-T, PH 7.4). Subsequently, the blots were incubated overnight at 4°C with one of the following antibodies: rabbit anti-p-AMPK (1:500; cat. no. ab23875; Abcam, USA) and rabbit anti-sirt1 (1:500; cat. no. ab220807; Abcam, USA). Membranes were washed three times for 5 min each time in TBS-T. HRP-coupled goat antirabbit secondary antibodies (1:1,000; Boster, Wuhan, China) diluted in TBS-T were then applied for 1 h. Membranes were washed three times in TBS-T for 5 min each time at room temperature. Immunoreactive signals were then visualized with the enhanced chemiluminescence solution (Bio-Rad, USA). Signal intensities were quantified by densitometric analysis using ImageJ software (Dental Diagnosis Science, San Antonio, TX).