Western blot analysis

All the tissues from individual rats were sampled for the analysis. In brief, the hippocampus of the rats was removed. Total protein was then extracted by homogenizing the hippocampus sample in ice-cold immunoprecipitation assay buffer with a protease inhibitor cocktail kit. The lysates were centrifuged and the supernatants were collected for measurements of protein concentrations using a bicinchoninic acid assay reagent kit.

After being denatured by heating at 95°C for 5 min in buffer, the supernatant samples containing 20 μg of protein were loaded onto 4-20% Mini-PROTEAN TGX gels and electrically transferred to a polyvinylidene fluoride membrane. The membrane was blocked in 5% nonfat milk in 0.1 % Tween-TBS buffer and was incubated overnight with respective primary antibody. The primary antibodies include: rabbit anti-p-mTOR /p-S6K1/P-4E-BP1 antibodies (1:200); rabbit anti-mTOR/S6K1/4E-BP1 antibodies (1:200-1:500); and rabbit anti-caspase-3 (cleaved) antibody (1:200). Next,the membranes were washed and incubated with an alkaline phosphatase conjugated anti-rabbit secondary antibody (1:500). All these primary and secondary antibodies were purchased from the Abcam Co. (Abcam, Cambridge, UK) and Santa Cruz Biotechology, Inc. (Santa Cruz, CA, USA). The immunoreactive proteins were detected by enhanced chemiluminescence. The bands recognized by the primary antibody were visualized by exposure of the membrane onto an X-ray film. The membrane was stripped and incubated with mouse anti-β-actin to show equal loading of the protein.Then, the film was scanned and the optical density of all protein bands was first analyzed using the Scion Image software, and values for densities of immunoreactive bands/β-actin band densities from the same lane were determined. Each of the values was then normalized to a control sample.