Ab104274

Total cell lysates were prepared from cells or brains using RIPA buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1% sodium deoxycholate, 1% Triton X-100, and 0.1% SDS) supplemented with protease inhibitors (Calbiochem) and incubated with Benzonase nuclease (Novagen) at room temperature for 5 min. After sonication for 30 sec, the lysates were centrifuged at 20,000 × g for 10 min at 4°C to remove insoluble debris. Proteins were separated on 10% SDS-PAGE gels and transferred to nitrocellulose membranes. The membranes were blocked with 2% FBS/0.5% skim milk in PBST (0.05% tween 20) and probed with various antibodies at 4°C overnight. The bands were visualized using an ECL detection system (GE Healthcare) and analyzed with a LAS4000 mini Luminescent Image Analyzer (GE Healthcare). The band intensity was quantified by using ImageQuant TL (GE Healthcare). The antibodies used were: NDUFA9 (1:250 Abcam ab14713), SDHA (1:1000 Molecular Probe A-11142), UQCRC2 (1:500 Abcam ab14745), ATP5α (1:1000 Molecular Probe A-11144), Drp1 (1:500 Abcam ab56788), Mfn1 (1:500 Abcam ab104274), Mfn2 (1:500 Abcam ab124773), VDAC1/Porin (1:800 Abcam ab15895), ATF-4 (1:1000 CST 11815), β-actin (1:1000 MBL M177-3).

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).

Equal amounts of protein (15 μg) were separated by SDS-PAGE on a 12% gel. The proteins were transferred onto a polyvinylidene difluoride membrane (BIO-RAD, #1704273, Hercules, CA). The membrane was incubated overnight with primary antibodies against total rodent OXPHOS (ab110413, Abcam), MT-NDI (ab181848, Abcam), OPA1 (ab42364, Abcam), DRP1 (ab56788, Abcam), MFN1 (ab104274, Abcam), MFN2 (ab56889, Abcam) and β-actin (sc47778, Santa Cruz Biotechnology) and incubated for 1 h with HRP-conjugated secondary antibodies (#32430 and #32460, Invitrogen or Thermo Scientific). The protein bands were detected using the enhanced chemiluminescent plus system.