*2.2. Western Blotting*

The protocol of western blotting used has been previously described [20]. In brief, hippocampal tissue was isolated on ice and homogenized in lysis buffer with a pellet mixer. The aliquot of hippocampal lysates was boiled for 5 min in Laemmli sodium dodecyl sulfate (SDS) sample buffer [60 mm Tris-Cl (pH 6.8), 2% sodium dodecyl sulfate, 10% glycerol, 4% β-mercaptoethanol, and 0.01% bromophenol blue]. A total of 10 μg of each SDS protein sample was loaded per lane, separated by 7.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to polyvinylidene difluoride membranes. Membranes were blocked using 4% Block Ace (Yukijirushi Ltd., Sapporo, Japan), incubated with the indicated primary antibodies, followed by incubation with horseradish peroxidase-conjugated secondary antibodies. Primary antibodies were rabbit anti-phospho-IRS1 [mouse Ser307/human Ser312 (mSer307/hSer312), mouse Ser612/human Ser616 (mSer612/hSer616), mouse Ser632/Ser635/human Ser636/Ser639 (mSer632/Ser635/hSer636/Ser639), mouse Ser1097/human Ser1101 (mSer1097/hSer1101)] (1:500; Cell Signaling Technology(CST)), rabbit anti-IRS1 (1:1000; CST), rabbit anti-phospho-Akt (Ser473) (1:1000; CST), rabbit anti-Akt (1:1000; CST), rabbit anti-phospho-p70S6K (Thr389) (1:1000; CST), rabbit anti-p70S6K (1:1000; CST), rabbit anti-phospho-AMPK (Thr172) (1:1000; CST), rabbit anti-AMPK (1:1000; CST), rabbit anti-phospho-GSK3β (Ser9)(1:1000; CST), rabbit anti-GSK3β (Ser9) (1:1000; CST), rabbit anti-phospho-JNK (Thr183/Tyr185) (1:1000; CST), rabbit anti-JNK (1:1000; CST), rabbit anti-phospho-aPKCζ/λ(Thr410/Thr403) (1:1000; CST), rabbit anti-aPKCζ/λ(1:1000; CST), and rabbit anti-β-tubulin (1:1000; CST). Immunodetection was performed with horseradish peroxidase-conjugated secondary antibodies (1:2000; CST) and chemiluminescence detection reagent Chemi-Lumi one L (Nacalai tesque, Kyoto, Japan) or the ImmunoStar LD (Fujifilm Wako Pure Chemical Corporation Japan, Osaka, Japan). The images were scanned using the Amersham Imager 680 (GE Healthcare UK Ltd., Little Chalfont, Buckinghamshire HP7 9NA, England ).
