Odyssey clx instrument system

Total proteins from the brains and Scn1a KO HT22 cells were prepared and extracted using the BCA Protein Extraction Kit (KGP2100, KeyGEN, Nanjing, China). Protein concentration was measured using the BCA Protein Assay Kit (KGP902, KeyGEN, Nanjing, China). Equal amounts of protein (50 μg per lane) were resolved on 8 or 10% sodium dodecyl sulfate (SDS) - polyacrylamide gel (SDS-PAGE), and then transferred onto 0.22 μm polyvinylidene fluoride (PVDF) membrane (Millipore, USA). When the protein transfer was completed, membranes were blocked with 5% non-fat milk for 1 h, followed by incubation overnight at 4°C with rabbit anti-SCN1A (1:500), p-mTOR (1:500), mTOR (1:500), Cleaved-Caspase3 (1:500), BAX (1:500), BCL-2 (1:500), or β-actin (1:1,000) primary antibody based on the validation results from the manufacturers. After incubation with primary antibody, membranes were washed with TBST three times for 5 min each time. β-actin served as internal references. Membranes were further incubated with the corresponding secondary antibody goat anti-rabbit IgG (1:1,000; LI-COR, USA) for 1.5 h. Quantification of bands was performed from optical density values using the Odyssey CLX instrument system (LI-COR, USA). All experiments were performed in triplicates.