Pgc 1α

First, 2 mL/well cells (about 2 × 10⁵) were seeded in 6-well plates and treated according to the protocol. Cells were collected and washed twice with PBS; then, they were resuspended in RIPA Lysis Buffer (Biosharp, Hefei, China) and supplemented with 1 mM of phenylmethanesulfonyl fluoride. Protein was extracted by centrifugation at 14,000× g for 15 min at 4 °C, and the concentration of protein was measured with a BCA protein assay kit (Thermo Scientific, Waltham, USA). Equal amounts of protein (80–150 µg) were separated by 10–20% SDS-PAGE gel and transferred to PVDF membranes at different electric currents according to the size of protein molecules. The membranes were blocked for 2 h in 5% non-fat milk dissolved with Tris-buffered saline containing 0.05% Tween-20 (TBST) at room temperature. Protein expression was detected using a primary antibody γ-H2A.X (1:1000, Abcam, Cambridge, MA, USA), BDNF (1:5000, Abcam, Cambridge, MA, USA), AMPKα (1:1000, CST, Danvers, MA, USA), PGC-1α (1:2000, Abcam, Cambridge, MA, USA), ULK2 (1:1000, CST, Danvers, MA, USA), SIRT1 (1:1000, CST, Danvers, MA, USA), β-actin (1:5000, Abcam, Cambridge, MA, USA), and horseradish peroxidase-conjugated anti-rabbit secondary antibodies (1:10,000, Abcam, Cambridge, MA, USA). A Quantitative analysis of a western blot was performed using Image-Pro Plus software (Media Cybernetics, Rockville, MD, USA).