Asc 001

Whole brain was homogenised in ice-cold buffer (150 mM NaCl, 50 mM Tris-HCl, 1% Nonidet P-40, 0.5% sodium deoxycholate and 0.1% SDS) containing protease inhibitors (Promega, Madison, WI, USA) and centrifuged at 10,000 g (30 min at 4°C). Supernatant was stored at −20°C. Antibodies were as follows: anti-PUM1 (1:1000, #12322, Cell Signaling Technology, Danvers, MA, USA), anti-PUM2 (1:1000, ab10361, Abcam, Cambridge, UK), anti-SCN1A (1:1000, ASC-001, Alomone Labs, Jerusalem, Israel), anti-SCN2A (1:1000, ASC-002, Alomone Labs), anti-SCN8A (1:1000, ASC-009, Alomone Labs), anti-GLUR2 (1:2000, AB1768-I, Merck, Darmstadt, Germany) and anti-β-actin (1:5000, ab8227, Abcam). Samples (25 µg of protein) were separated by SDS-PAGE, and protein was transferred to a polyvinylidene difluoride membrane (GE Healthcare). After blocking [0.5% bovine serum albumin (BSA) and 0.05% Tween 20 in Tris-buffered saline (TBS-T)], membrane was incubated overnight (4°C) in primary antibody diluted in 0.5% BSA in TBS-T. Membranes were incubated with horseradish peroxidase-conjugated secondary antibody (1:2500, #7074, Cell Signaling Technology) in 0.3% BSA in TBS-T, and blots were developed with an Enhanced Chemiluminescent Detection Kit (Pierce, Rockford, IL, USA). Protein band density was measured using ImageJ (National Institutes of Health, Bethesda, MD, USA).

Protein extraction from rat brains was performed at P13 using an ULTRARIPA kit for lipid rafts (F015; BioDynamics Laboratory Inc.); the reagent solution contained 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% NP-40 alternative, 0.1% SDS, and 0.5% sodium deoxycholate. Proteins were separated using SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore). The membranes were blocked with 5% nonfat skim milk overnight at 4°C and incubated in primary antibody solution containing rabbit anti- voltage-gated sodium channel alpha subunit 1 (Na_V1.1) (ASC-001, 1:200; Alomone Labs) and mouse anti-β-actin (A1978, 1:1000; Sigma-Aldrich) for 1 h at room temperature and sequentially incubated with secondary antibodies containing anti-Rb IgG HRP (AP307P, 1:2000; Millipore) and anti-mouse IgG HRP (AP308P, 1:2000; Millipore) for 1 h at room temperature. The signal was detected using Chemiluminescence HRP Substrate (Takara Bio) on the LAS3000 Mini apparatus (FUJI FILM). Semiquantitative analysis of the signal was performed using ImageJ software (National Institute of Health).