Western blot analysis

After treatment, primary cortical neurons were washed with PBS and extracted with lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) containing protease inhibitor cocktail (Hoffman-La Roche Ltd., Basel, Switzerland) and 1 mM phenylmethylsulfonyl fluoride for 30 minutes on ice. The total protein lysates were separated by SDS-polyacrymlamide gel electrophoresis and analyzed by Western blotting with primary antibodies (Akt, phosphorylated Akt [p-Akt], mTOR, phosphorylated mTOR [p-mTOR], ribosomal protein S6 kinase [p70S6K], phosphorylated p70S6K [p-p70S6K; 1:1,000] and β-actin [1:5,000]) from Cell Signaling (Danvers, MA, USA) overnight at 4°C, followed by horseradish peroxidase-conjugated secondary antibody (1:10,000) for 2 hours at room temperature. The results were analyzed by using ImageJ software (National Institutes of Health). The mean density of each band was normalized to the β-actin signal in the same sample and averaged.