2.2. Whole brain lysate preparation and western blotting

Mice were sacrificed by CO₂ inhalation and rapidly decapitated. Their brains were removed, dissected for specific brain regions (hippocampi, cortexes and cerebellums) and stored in −80°C. The dissected brain regions per mouse were homogenized in ice‐cold homogenization buffer (solution A) containing (in mM): 320 sucrose, 1 NaHCO₃, 1 MgCl₂, 0.5 CaCl₂, 0.4 HEPES (pH 7.4) and Halt protease inhibitors (Thermo Fisher Scientific) as previously described.54 After centrifuging 1400g for 10 minutes at 4°C, the homogenate supernatant (S1) was separated from insoluble tissue and nuclear pellet (P1). The S1 fraction was then centrifuged at 13800g for 10 min at 4°C. The supernatant (S2) was removed, and the remaining pellet (P2 membrane fraction) was resuspended in ice‐cold solution B containing (in mM): 160 sucrose, 6 Tris‐HCl, 0.5% Triton‐X (pH 8.0) and Halt protease inhibitors. The S2 fraction is enriched with cytosolic soluble proteins. The P2 fraction is enriched with transmembrane proteins and membrane‐bound proteins. BCA assay (Pierce) analysis was performed to determine protein concentrations across samples, which were subsequently normalized to 0.5 mg/mL in Solution A (pH 7.4). The S1, S2 and P2 fractions were stored at −80°C until use. Hippocampal S2 and P2 fractions were immunoblotted with antibodies for K_v7.2 (1:200, Neuromab cat# 73‐079), K_v7.3 (1:200, Alomone cat# APC‐051) and GAPDH (1:1000, Cell Signaling) as described.54 After incubating in HRP‐conjugated secondary antibodies, the blots were visualized with enhanced chemifluorescence substrate (ECL, Thermo Fisher Scientific), and developed with a Konica SRX‐101A film processor.