Total lab quant v11

Western blot analysis was performed as described previously [26] . In brief, the protein concentration was measured using a bicinchoninic acid protein assay kit (YTHXBio, China). The extracted proteins were then separated by electrophoresis using a 12% sodium dodecyl sulphate polyacrylamide gel. The proteins were transferred to polyvinylidene uoride membranes (Millipore, USA) and blocked using Tris-buffered saline with Tween buffer containing 3% BSA. The membranes were incubated with primary antibodies against Bax (1:500), phosphorylated AMPK (Thr 172) (1:1000), total AMPK (1:5000) (all from Cell Signaling Technology, Danvers, MA, USA), Bcl-2 (1:5000; Abcam), and GADPH (1:10000; YTHXBio, China) at 4°C overnight. The membranes were washed three times and then incubated with the horseradish peroxidase-conjugated secondary antibody (1:10,000) at 37°C for 40min. Finally, the bands were detected by an enhanced chemiluminescence kit (Millipore, USA) and the results were quanti ed using Total Lab Quant V11.5 (Newcastle upon Tyne, UK). The levels of Bcl-2/Bax (anti-/pro-apoptotic) proteins and AMPK-p/AMPK proteins were also normalized to that of GAPDH.