Membrane Protein Deglycosylation and Western Blotting

Brain membrane proteins were prepared from P60 to 90 mice as described.^{41 (link)} Complete protease inhibitors (Roche Diagnostics) were added to all solutions at twice the recommended concentration to minimize protein degradation. Deglycosylation of membrane protein samples, where indicated, was performed using PNGaseF (New England BioLabs Cat. #P0704S) as previously described.^{17 (link)} For western blotting, 50–80 µg aliquots of membrane protein were separated by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and processed for western blotting with anti-β1_intra antibody (1:1000) (Cell Signaling Technologies 13950) or anti-Na_V1.1 Na⁺ channel antibody (K74/71) (1:200) (NeuroMab 75-023) as previously described.^{22 (link)} Anti-α-tubulin (1:1000) (Cedar Lane) or anti-transferrin receptor (TfR) (1:200) (Invitrogen H68.4 13-6800) antibodies were used as loading control, as indicated. Immunoreactive bands were detected using Supersignal West Dura Extended Duration Substrate (Therma Scientific #34076) and imaged using an iBrightFL1000 system (Invitrogen). Immunoreactive signals for the deglycosylated bands were quantified using iBright analysis software (Invitrogen) and normalized to the level of α-tubulin for each sample.