Protein Expression Analysis in Hippocampal Slices

Hippocampal slices were flash-frozen on dry ice and sonicated as previously described (Ma et al., 2013 (link)). Samples containing equal amounts of protein lysate were loaded on 4 – 12% Tris-glycine SDS-PAGE gels for standard gel electrophoresis. Membranes were probed overnight at 4°C using primary antibodies for the following antibodies: Kv4.2 (1:5000; Alomone Labs); GluA1 (1:1000; Cell Signaling); p-mTOR (Ser2448) (1:1000; Cell Signaling); mTOR (1:1000; Cell Signaling); p-p70S6K (Thr389) (1:1000; Cell Signaling); p70S6K (1:1000; Cell Signaling); p-4EBP1(Thr37/46) (1:1000; Cell Signaling); 4EBP1 (1:1000; Cell Signaling); p-Akt (Ser473) (1:1000; Cell Signaling); Akt (1:1000; Cell Signaling); p-GSK3β (Ser9) (1:1000; Cell Signaling); GSK3β (1:1000; Cell Signaling); p-eIF2α (Ser51) (1:1000; Cell Signaling); eIF2α (1:1000; Cell Signaling); p-eEF2 (Thr56) (1:1000; Cell Signaling); eEF2 (1:1000; Cell Signaling); eEF1A (1:5000; Millipore); p-AMPKα (Thr172) (1:1000; Cell Signaling); AMPKα (1:1000; Cell Signaling); GAPDH (1:10,000; Cell Signaling). Protein bands were visualized using chemiluminescence (Clarity™ ECL; Biorad) and the Biorad ChemiDoc™ MP imaging system. Densitometric analysis was performed using ImageJ. Data were normalized to GAPDH (for total proteins analysis) or relevant total proteins (for phospho proteins analysis) unless otherwise specified.