Anti coxiv 3e11

Proteins were separated by SDS–PAGE under reducing conditions and transferred to poly(vinylidene fluoride) membranes. Membranes were blocked with 5% nonfat milk in phosphate-buffered saline with 0.1% Tween 20 (Sigma-Aldrich, P9416; PBST). The blots were incubated overnight at 4 °C with primary antibodies. Immunoblot analysis was performed using an enhanced chemiluminescence procedure (GE Healthcare, RPN2106), and western blot images were captured using the ChemiDoc™MP system (Bio-Rad), Image Lab version 6.1.0. Densitometric analysis was performed using ImageJ software (National Institutes of Health). The following antibodies were used: Cell Signaling Technology: anti-COXIV (3E11, #4850), 1:1000; anti-VDAC1 (D73D12, #4661), 1:1000; anti-Tomm20 (D8T4N, #42406), 1:1000; anti-GAPDH (14C10, #2118), 1:10000; anti-Cytochrome c (136F3, #4280), 1:1000; anti-LC3B (#2775), 1:1000; Novus Biologicals: anti-Dhps (NBP1-82648), 1:1000; BD Biosciences: anti-Eif5a (611976), 1:1000; Sigma-Aldrich: anti-Dohh (HPA041953), 1:1000; Millipore: anti-Hypusine (ABS1064), 1:1000; anti-puromycin (12D10, #MABE343), 1:1000; Abcam: anti-Tfam (ab131607), 1:1000; anti-PGC1α (ab54481), 1:1000; anti-TFEB (ab2636), 1:1000; and Atp5a, Uqcrc2, Sdhb, and Ndufb8 were detected using Total OXPHOS Rodent WB Antibody Cocktail (ab110413), 1:1000. Primary antibodies were diluted in 1% BSA containing PBST.

The heptamethine cyanine dye IR 783 (2‐[2‐[2‐chloro‐3‐[2‐[1,3‐dihydro‐3,3‐dimethyl‐1‐(4‐sulfobutyl)‐2H‐indol‐ 2‐ylidene]‐ethylidene]‐1‐cyclohexen‐1‐yl]‐ethenyl]‐3,3‐dimethyl‐1‐(4‐sulfobutyl)‐3H‐indolium) was purchased from Sigma‐Aldrich (115970‐66‐6). MitoTracker Red CMXRos (M7512) was purchased from Life Technologies. The following antibodies were used: anticytochrome c (Cyto C, sc‐13156), anti‐OPA1 (sc‐393296), anti‐MFF (sc‐398617), anti‐Mfn1 (sc‐166644) and anti‐Fis1 (SC‐376447) were from Santa Cruz Biotechnology; anti‐Drp1 (611113) was from BD Biosciences; anti‐actin (065M4837V) was from Sigma‐Aldrich; anti‐cleaved‐caspase‐3 (C‐Caspase‐3, 9661), anti‐Cox IV (3E11) and anti‐cleaved‐poly ADP‐ribose polymerase (C‐PARP, 5625) were from Cell Signaling Technology.