Western blotting

Hippocampal tissue samples were homogenised in 1× PBS using an Ultra-Turrax TP18-10 (Janke & Kunkel KG) and then solubilised in lysis buffer (10 mM Tris pH 8.0, 1 mM EDTA, 1% Triton-X, 0.1% sodium-deoxycholate, 0.1% SDS, 140 mM NaCl) supplemented with phosphatase and protease inhibitors for 30 min on ice. To remove insoluble material, the protein extracts were centrifuged for 10 min at 20,000×g at 4 °C. The supernatants were then stored at −20 °C until further processing. An identical lysis solubilisation procedure was executed for N2a cells. Protein content was measured using the Pierce BCA Protein Assay Kit according to manufacturer's instructions. Proteins were electrophoretically separated on density gradient polyacrylamide gels and transferred onto polyvinylidene difluoride membranes (GE Healthcare; Uppsala, Sweden) using semi-dry transfer in TransBlot Turbo (Bio-Rad).

Following transfer, the membranes were blocked in PBS containing 0.05% Tween 20 (PBS-T) and 5% non-fat dry milk for 1 h at room temperature. Membranes were subsequently incubated with primary antibody (Supplemental Table S3) in 5% non-fat dry milk in PBS-T overnight at 4 °C. After primary antibody incubation, the membranes were washed 5× in PBS-T and incubated in secondary horseradish peroxidase (HRP)-conjugated anti-mouse or anti-rabbit antibody (Supplemental Table S3) for 1 h at room temperature in PBS-T. The membranes were then washed 5× with PBS-T and visualised by enhanced chemiluminescence using a ChemiDoc MP (Bio-Rad). As a loading control, membranes were probed with primary mouse anti-tubulin or mouse anti-actin for 1 h at room temperature and sequentially incubated with HRP-conjugated anti-mouse antibody for 1 h at room temperature in PBS-T. Protein expression was then detected as described above.