For immunoblotting of samples in blue native gels, protein complexes from 3–12% precast Bis–Tris Native PAGE gels (Life Technologies) were transferred to polyvinylidene difluoride membranes (Bio-Rad). For immunoblotting of denatured samples in whole tissue lysates, thoraxes were homogenized in radioimmunoprecipitation assay buffer (150-mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, and 50-mM Tris-HCl, pH 8) supplemented with Halt protease inhibitors (Pierce), resolved on mini-PROTEAN TGX stain-free gels from Bio-Rad, and transferred to polyvinylidene difluoride membranes. In both instances (native and nonnative gels), the membrane was subsequently blocked in 5% (wt/vol) nonfat dry milk in TBS for 30 min and incubated in the appropriate primary antibody dissolved in 2% BSA and 0.1% Tween 20 in TBS (TBST) overnight at 4°C. Following the overnight incubation, the blot was rinsed four times for 10 min each in 0.1% TBST, blocked for 30 min in 5% (wt/vol) nonfat dry milk in TBST, and incubated for 2 h with the appropriate HRP-conjugated secondary antibody dissolved in 2% BSA and 0.1% TBST. After incubation in the secondary antibody, samples were rinsed four times for 10 min each in 0.1% TBST. Immunoreactivity was detected by ECL and analyzed by a ChemiDoc Gel imaging system from Bio-Rad. Primary antibodies used were anti-NDUFS3 (Abcam, ab14711), anti-ATPsynβ (Life Technologies, A-21351), anti-VDAC1/Porin (Abcam, ab14734), anti–cytochrome C (Abcam, ab13575), anti-Hsp60 (Cell Signaling Technology, 4869S), anti-Hsp90 (Cell Signaling Technology, 4874S), and the rabbit polyclonal antibodies generated by Biomatik. Secondary antibodies used were goat anti-rabbit HRP (Pierce, PI31460) and goat anti-mouse HRP (Pierce, PI31430). There are major discrepancies between the migration behavior of membrane and soluble protein markers. Accordingly, estimating the sizes of membrane proteins such as OXPHOS complexes or AIs on blue native gels using standard soluble protein markers produces spurious results. Consequently, we chose not to estimate the sizes of proteins on blue native gels using standard protein markers. OXPHOS complexes and AIs were assessed based on their known constituent protein subunits.