The total protein (35 (link)) and surface protein (plasma membrane protein) (36 (link)) of the hippocampus were extracted using the Protein Extraction Kit (Invent Biotechnologies, SD-001/SN-002; SM-005). The bicinchoninic acid (BCA) kit (Beyotime, P0012S) was used to evaluate protein concentration. Protein samples (20 µg protein/lane) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose (NC) membranes (Merck Millipore Ltd) by wet transfer (Bio-RAD) for western blot. The NC membranes were blocked by 5% Bovine Serum Albumin (BSA) and then incubated with a primary antibody at 4 ℃ overnight. The primary antibodies used included anti-KIF5A antibody 1:1,000 (SANTA, sc-376452), anti-GluR2 antibody 1:1,000 (BOSTER, PB9205), and anti-GABAaR β2+3 antibody 1:500 (Bioss, bs-12066R).
Next, the NC membranes were incubated with horseradish peroxidase (HRP)-conjugated Affinipure goat anti-rabbit immunoglobulin G (IgG) (H+L) 1:8,000 (Proteintech, SA00001-2) and HRP-conjugated Affinipure goat anti-mouse IgG (H+L) 1:8,000 (Proteintech, SA00001-1) at room temperature for 1 hour. After the NC films were incubated with the Omni-ECL™Femto Light Chemiluminescence Kit (EpiZyme, SQ201) for 1–2 min, the ChemiScope6000 gel imaging system was used to observe and photograph the results.
Next, the NC membranes were incubated with horseradish peroxidase (HRP)-conjugated Affinipure goat anti-rabbit immunoglobulin G (IgG) (H+L) 1:8,000 (Proteintech, SA00001-2) and HRP-conjugated Affinipure goat anti-mouse IgG (H+L) 1:8,000 (Proteintech, SA00001-1) at room temperature for 1 hour. After the NC films were incubated with the Omni-ECL™Femto Light Chemiluminescence Kit (EpiZyme, SQ201) for 1–2 min, the ChemiScope6000 gel imaging system was used to observe and photograph the results.