Cell lysis, protein quantification, and western blotting

Cells were lysed in radioimmunoprecipitation assay buffer (RIPA, tris-hydrochloric acid (MP Biomedicals, Santa Ana, CA), sodium chloride (Fisher), sodium deoxycholate (Fisher), NP-40 (Thermo Fisher Scientific, sodium dodecyl sulfate (SDS, MP Biomedicals), and EDTA (MP Biomedicals)) with 5% protease inhibitor (Roche, Basel, CH) and lysate stored at − 20 °C. Samples run on 12% tris/glycine gels (Bio-Rad, Hercules, USA) were transferred to nitrocellulose membranes, blocked with 2% skim milk, and incubated with primary antibodies overnight at 4 °C. Membranes were washed and incubated with secondary antibody at room temperature with Clarity™ Western ECL Blotting Substrate (Bio-Rad) added before imaging on a Bio-Rad ChemiDoc™ MP System. Blots were analyzed using quantitative densitometry on Image Lab™ (Bio-Rad 5.2) and normalized to tubulin as a loading control following normalization of data on different blots to a positive control spinal cord lysate that was loaded on each blot run; this enabled blot to blot comparisons when run in an identical manner and using the same exposure times.