Preparation of membrane-enriched extracts and western blotting

Membrane-enriched protein fractions (P2) were obtained from cells and brain tissues as previously described.62 Protein concentration was determined by Bradford assay (Bio-Rad), and part of the preparation was solubilized in denaturing conditions as described previously.11^,63 Protein samples were mixed with 4× urea-EDTA buffer, resolved onto 7.5% SDS-polyacrylamide gels, and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% BSA and 0.05% Tween 20 in Tris-buffered saline (TBS), followed by overnight incubation at 4°C with the following primary antibodies: rabbit polyclonal anti-Nav1.1 (Alomone Labs Cat# ASC-001, 1:500) and rabbit monoclonal (14C12) anti-GAPDH (Cell Signaling Technology, Cat# 2118S, 1:5,000) diluted in 2.5% BSA, 0.05% Tween 20, and 0.01% azide in TBS. Immunolabeled protein bands were detected by using an anti-rabbit immunoglobulin G (IgG) horseradish peroxidase (HRP) conjugate (GE Healthcare, Cat# NA934V, 1:10,000) and an enhanced chemiluminescence system (Lumigen ECL Ultra TMA-6, Lumigen, Cat# TLA-100). Images were acquired with a ChemiDoc system (Bio-Rad).