Phospho s6 ser240 244

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.

Retinas were dissected out from 4% paraformaldehyde (PFA)-fixed eyes and washed extensively in PBS before blocking in staining buffer (10% normal goat serum and 2% Triton X-100 in PBS) for 30 min. Mouse or rabbit antibodies for neuronal class ß-III tubulin (clone Tuj1, 1:500; Covance), rat HA (clone 3F10, 1:200; Roche), phospho-S6-Ser240/244 (1:200, #5364; Cell Signaling), phospho-AKT-Ser473 (1:200, #4058; Cell Signaling), pan AKT (1:200, #2920; Cell Signaling), and phospho-GSK-3β (Ser9) (1:100, #9323; Cell Signaling) were diluted in the same staining buffer. RBPMS guinea pig antibody was made at ProSci, CA, according to publications^{31 (link),32 (link)}. Floating retinas were incubated with primary antibodies overnight at 4 °C and washed three times for 30 min each with PBS. Secondary antibodies (Cy2, Cy3, or Cy5 conjugated) were then applied (1:200; Jackson ImmunoResearch) and incubated for 1 h at room temperature. Retinas were again washed three times for 30 min each with PBS before a cover slip was attached with Fluoromount-G (SouthernBiotech).