Densitometry

The expression levels of p-AMPK, p-P65NF-κB, p-P38MAPK, Bace1, and APP were analyzed by Western blotting. The protein samples were heated at 100 °C for 5 min with a loading buffer containing 0.125 M Tris-HCl (pH 6.8), 20% glycerol, 4% SDS, 10% β-mercaptoethanol, and 0.002% bromophenol blue. The samples were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene (PVDF) membranes. The membranes were incubated with 3% bovine serum albumin (BSA) in Tris-buffered saline with Tween (TBST) (10 mmol/L Tris at pH 7.5, 150 mmol/L NaCl, 0.05% Tween-20) and probed with corresponding primary antibodies (anti-p-AMPK, anti-p-P38MAPK, anti-p-P65NF-κB, anti-BACE1, and anti-APP; Cell Signaling Technology, Boston, USA) at 4 °C overnight. After incubation with horseradish peroxidase-coupled secondary antibodies for 2 h at room temperature, the protein bands were quantified by densitometry (Syngene, UK).