Sc 5263

Cell pellets were lysed in RIPA buffer containing 1× Halt^TM protease inhibitor cocktail (78437, Thermo) and supernatants collected after 10 min centrifugation at 12000 × g for SDS-PAGE. Total proteins were transferred to PVDF membranes and blotted with the antibodies against FIS1 (ab71498, Abcam), MFN1 (ab104274, Abcam), β-Actin (ab8227, Abcam), Caspase-3 (9665, Cell Signaling), Caspase-8 (sc-5263, Santa Cruz), Caspase-9 (sc-8355, Santa Cruz), HSP70 (TA309356, Origene) and Total OXPHOS Human WB Antibody Cocktail (ab110411, Abcam).

Western blotting was performed as previously described [29 (link),30 (link)]. In short, the cell lysates were denatured in a sample buffer containing sodium dodecyl sulfate (SDS), and equal amounts of total protein were separated on 8%–15% SDS-poly-acrylamide gels and transferred to nitrocellulose membranes. After blocking with 5% nonfat dry milk, the membranes were incubated overnight at 4 °C with the primary antibodies as indicated. The following antibodies were used: against active-caspase-3 (sc-271028), active-caspase-8 (sc-5263), active-caspase-9 (sc-133109), cleaved cleaved-poly adenosine diphosphoribose polymerase 1 (PARP-1) (sc-56196), cytochrome c (sc-13156), cytochrome c oxidase IV (COX IV) (sc-376731), Bcl-2 (sc-492), and Bax (sc-526) were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Anti-â-actin was purchased from Sigma Chemical Co. (St. Louis, MO, USA), and these antibodies against LC3, Atg5/12 conjugate, and Beclin-1 were purchased from Novus Biological Inc (Littleton, CO, USA). The following day, the membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies, and detection was performed using enhanced chemiluminescence (ECL) reagent (Amersham Life Science).