Immunoblotting Procedure for Hippocampal Protein Analysis

Immunoblotting has been described previously [10 (link),11 (link)]. A bicinchoninic acid (BCA; Thermo Fisher Scientific, Waltham, MA, USA) protein assay using bovine serum albumin (BSA; Sigma, St. Louis, MO, USA) as a standard was used to determine protein concentrations. Hippocampal protein samples were heated for 5 min to 95 °C (GLT-1, actin, CP13, MC1, and PHF1) or not heated (vGLUT1, PSD95, Tau-5, HT7, synaptophysin, actin, GSK3β, pGSK3β) and loaded on 10% hand-cast sodium dodecyl sulfate (SDS)-page gels. After transfer, membranes were blocked for 1 h at room temperature (∼23 °C) and then incubated with a primary antibody (see Table 1 for list of antibodies) directed against the protein of interest overnight at 4 °C. The next day, membranes were incubated with the appropriate biotinylated or horseradish peroxidase conjugated secondary antibody for 1 h at room temperature (∼23 °C). Blots were then incubated with SuperSignal West Pico chemiluminescent substrate (Thermo Scientific, Waltham, MA, USA) or Novex AP chemiluminescent substrate (Invitrogen) for 5 min and visualized using Fluorchem E imager (Cell Biosciences, Preston VIC, Australia).