Mice were sacrificed by cervical dislocation. mPFC or hippocampi from both sides of the brain were rapidly isolated. Hippocampi were then divided into dorsal and ventral parts along the longitudinal axis. The tissues were placed into liquid nitrogen followed by long storage at −80°C. For Western blotting analysis, samples were removed from the freezer and homogenized in a lysis buffer containing 20 mM tris-Cl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 5 mM EDTA, 3 mM NaF, 1 mM sodium orthovanadate, 10% glycerol, and complete protease inhibitor set (Sigma-Aldrich, St. Louis, MO). The lysates were vortexed for 20 s, incubated on ice for 40 min, and centrifuged at 13,000 rpm for 15 min. The supernatants were transferred to sterile 1.5-ml Eppendorf tubes for subsequent experiments. Protein concentrations were determined by the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA). Twenty micrograms of total protein extract per lane was resolved using denaturing SDS–polyacrylamide gel electrophoresis gels and transferred to polyvinylidene difluoride filters by electroblotting. The filters were incubated overnight at 4°C with appropriate primary antibodies. The following primary antibodies were used: goat anti-ASIC1a (1:1000; Santa Cruz Biotechnology, catalog no. sc-13905) and mouse monoclonal anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (1:1000; KangChen, catalog no. KC-5G4). Secondary antibodies conjugated to horseradish peroxidase were added to the filters and then visualized in enhanced chemiluminescence solution. The visualization was performed via the ImageQuant LAS 4000 Mini Molecular Imaging System (GE Healthcare Life Sciences), and the Quantity One software [National Institutes of Health (NIH)] was used for the analysis of band intensity.

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