Anti nav1.1 antibody

The hippocampi were extracted and homogenized as previously described (Nissenkorn et al., 2019 (link)). Briefly, 0.45–0.7 mg of tissue was homogenized in 0.32 M sucrose supplemented with protease inhibitors (Sigma-Aldrich, St. Louis, MO, USA), 1 mM EDTA and 1 mM PMSF, pH 7.4. Crude membrane preparation was produced by centrifugation at 27,000 × g for 75 min. The pellet was solubilized in 150 mM NaCl, 2% Triton X-100, 25 mM Tris, supplemented with protease inhibitors, 1 mM EDTA and 1 mM PMSF, pH 7.4. 50 μg aliquots of total protein were separated on Tris-acetate gel (6%) and transferred onto PVDF membrane. After overnight blocking in 5% non-fat dry milk in Tris-buffered saline (TBS), the membrane was incubated overnight with anti-Na_V1.1 antibody (1:200, Alomone Labs, Jerusalem, Israel; Catalog# ASC-001) or anti-calnexin (1:2,000, Stressgen Biotechnologies, San Diego, CA, USA), followed by 2 h incubation with HRP-conjugated goat anti-rabbit antibody (1:10,000, Sigma-Aldrich, St. Louis, MO, USA). The signal was visualized by chemiluminescent detection using ECL.

Cells were homogenized in 0.32M sucrose supplemented with protease inhibitors (Sigma-Aldrich, St. Louis, MO, USA), 1mM EDTA and 1mM PMSF, pH 7.4. Crude membrane preparation was produced by centrifugation at 17,000 x g for 90 min. The precipitate (pellet) was solubilized in 150 mM NaCl, 2% Triton X-100 supplemented with protease inhibitors, 1mM EDTA and 1mM PMSF, pH 7.4. A 35 mg aliquot of total protein was separated on Tris-acetate gel (6%) and transferred onto PVDF membrane. After blocking with 5% w/v nonfat dry milk in TBST, the membrane was incubated with anti-Na_V1.1 antibody (1:200, Alomone Labs, Jerusalem, Israel; catalog number ASC-001) or Alpha 1 Na⁺/K⁺ ATPase (1:200, Alomone Labs, Jerusalem, Israel; catalog number ANP-001), followed by incubation with HRP-conjugated goat anti-rabbit antibody (1:10,000, Sigma-Aldrich, St. Louis, MO, USA). The signal was visualized by chemiluminescent detection using the ECL Detection System.