Western blot and co-immunoprecipitation

For Western blot, proteins on an SDS gel were transferred onto nitrocellulose membranes, which were blocked by 5% milk in TBS, and then incubated with the primary antibody diluted in TBST (TBS + 0.1% Tween-20). Signal development was done using alkaline phosphatase conjugated secondary antibodies (Pierce) and the NBT/BCIP substrate (Life Technologies). The primary antibody and dilution used: α-MCU (Sigma, HPA016480, 1:2000), α-EMRE (Santa Cruz, 86337, 1:400), α-FLAG (Sigma, F1804, 1:4000), α-V5 (Life Technologies, 46–0705, 1:5000), α-Cyt-C (Santa Cruz, 13156, 1:1000), α-β-actin (Santa Cruz, 69879, 1:500), α-Letm1 (Abcam, 55434, 1:2000). Monoclonal anti-1D4 and -C8 antibodies were produced in house.

All co-IP experiments were performed at 4 °C. Transfected HEK 293 cells were grown in a 10-cm dish to confluency, were harvested, and then lysed in 1-mL solubilization buffer (SB, 100 mM NaCl, 20 mM Tris, 1 mM EGTA, 25 mM DDM, pH 7.5-HCl), supplemented with an EDTA-free protease inhibitor cocktail (cOmplete Ultra, Roche). The cell lysate was clarified by centrifugation, and a small portion of the sample was taken for whole cell lysate analysis. Antibody-conjugated Sepharose beads (25 μL) were added, and after 1 h, the beads were collected on a mini column, washed with 2-mL SB, and eluted with 200-μL SDS-gel loading buffer for Western blot. Antibody affinity gel used: FLAG (Sigma, A2220), V5 (Sigma, A7345). 1D4 and C8 affinity gels were produced using 20-mg 1D4 or C8 antibody per 1-g Sepharose 4B (GE Healthcare).