Mitochondria cross-link and mitochondrial DLP1 complex assay

M17 cells were seeded into 6-well plates for 24 h. After a wash with PBS, cells were incubated with HBSS containing 0.5 mM dithiobismaleimidoethane (DTME, from freshly prepared stocks in DMSO) for 5 min at 37 °C. The HBSS-DTME was removed and the mitochondrial fraction was isolated. Mitochondria were lysed in buffer containing 60 mM Tris pH 6.8 and 0.2% SDS. Insoluble debris was removed by centrifugation (10 min at 14,000 g) and cleared lysates were layered onto 1 ml of a 300-mM sucrose cushion and subjected to ultracentrifugation (30 min at 270,000 g). These 270,000 g pellets were dissolved in SDS sample buffer containing 5% β-mercaptoethanol to cleave the cross-linker and were analysed by SDS-PAGE.