Molecular Mechanisms of Cognitive Function

Rats were sacrificed at pre-determined time points for Western blot analysis of mTOR pathway activation. Standard methods were used as described previously [7 (link)]. In brief, total proteins extracted from temporal neocortex and hippocampus were separated by sodium dodecyl sulfate polyacrylamide electrophoresis and transferred to nitrocellulose membrane. Neocortex and hippocampus were assayed, in correlation with the behavioral studies on cognitive function. After blocking with 5% skim milk, the membranes were incubated with the primary antibodies: phospho-Akt (Ser473,) phospho-mTOR (Ser2478) and phospho-S6 (Ser240/244) (1:1,000; Cell Signaling Technology, Beverly, MA, U.S.A.) followed by peroxidase conjugated anti-rabbit secondary antibody (1:5000, pierce, Rockford, IL). Protein bands were visualized with enhanced chemiluminescence reagent (Pierce). The membranes were reprobed and incubated with the rabbit Akt, mTOR and S6 antibody (1:1,000; Cell Signaling Technology). After measurement of the intensity of each lane in each blot by ImageJ (NIH, Bethesda, MD, U.S.A.), the ratio of p-S6 to total S6 was calculated and normalized to the control group. Each experiment was conducted at least three times.