In vitro autophagic flux

The procedure was previously reported by Gomez-Sanchez et al. (2015). Primary mouse SCs were grown in culture for 4 d before being treated with 15 mm ammonium chloride for 3 h. Cells were collected for Western blot (WB) and lysed in RIPA lysis buffer; 5 µg of protein was separated by SDS-PAGE and probed for rabbit anti-LC3B and rabbit anti-GAPDH in 5% BSA/TBST. Autophagic flux was calculated by normalizing LC3II density to GAPDH density, then subtracting the untreated group from the treated group.