Ab151229

For J2 immunofluorescence on larvae brain, tissues were dissected in PBS, fixed for 5 minutes in 4% formaldehyde, and washed for 5 minutes. Larvae brain were then permeabilised for 2 hours in 0.5% Triton X-100 in PBS, and saturated for 1 hour in 0.5% BSA, 0.1% Tween 20 in PBS (PBTB). Primary antibodies anti-dsRNA (Scicons J2: anti-dsRNA/100105500) and anti ATP synthase subunit 5a (Abcam ab151229) were used at 1:200 in PBTB for overnight at 4°C, and later washed for 1 hour in 0.1% Tween in PBS (PBTW). Secondary antibodies (Molecular Probes, IgG,568, A-11031, and Life Technologies, Alexa Fluor488, A-11008) were used at 1:500 for 2 hours and washed for 1 hour in PBTW. Preparations were mounted in Vectashield/DAPI (Vector). A LSM880 Zeiss confocal microscope was used for imaging.
Mitochondria of the ventral nerve cord were visualised by crossing dmpnpase^KO, dmsuv3^KD, dmlrpprc1^KD,dmmtpap^KO, or dmpnpase^OE/dmsuv3^OE flies to previously generated UAS-mit::dendra2 flies (w;elav-gal4,uasmit::dendra2;) [54 (link)]. For in situ detection of mit::dendra2, living larval central nervous systems were rapidly dissected, mounted into PBS, and immediately imaged, using a LSM880 Zeiss confocal microscope.

Differentially abundant proteins of interest were examined by immunoblotting of pools of protein extracts from all experimental groups and controls from Parts I and II. Protein (10 µg per pool) was fractionated on 12% Criterion TGX Stain-Free gels (Bio-Rad). Gel images were acquired with the Chemidoc system (Bio-Rad) to document equal protein loading among samples. Then, proteins were electrotransferred onto nitrocellulose membranes and probed with following primary antibodies: anti-ATP5F1A (1:1000; #ab151229, AbCam), anti-SDHB [21A11AE7] (1:500; #ab14714, AbCam), anti-PGA3 (1:1000; #PA5-49728, Thermo Fisher Scientific), anti-pepsinogen II/PGC (1:500; #ab135862, AbCam), anti-PDIA3 (internal region; 1:1000; #ABIN3187755, Antibodies-online; Aachen, Germany), anti-GSTP1 (1:1000; #PA5-29558, Thermo Fisher Scientific), and anti-PSME1 (1:1000; #ab14714, AbCam). Primary antibodies were detected with HRP-conjugated goat anti-rabbit and anti-mouse IgG Fc fragment antibodies (1:10,000 dilution; Bethyl Laboratories, Montgomery (TX), USA) and Clarity Western ECL Substrate (Bio-Rad). Blots were imaged with the Chemidoc system.

IF primary: rabbit anti-CENP-A (Active Motif, #39713, 1:1000); guinea pig anti-CENP-C^{31 (link)} (1:1000); rabbit anti-ATPsyn-α (Abcam #ab151229, 1:100); mouse anti-ATPsyn-β (Abcam #ab14730, 1:200); goat anti-ATPsyn-γ (Abcam #ab190310, 1:200); rat anti-mCherry (Chromotek 5F8 1:500); mouse anti-tubulin (Sigma DM1A, 1:100); guinea pig anti-MEI-S332^{32 (link)} (gift from T. Orr-Weaver 1:500) and rabbit anti-GFP (Santa Cruz SC-8334). Guinea pig anti-ATPsyn-βlike antibodies (1:200) were generated by co-injection of two KLH and BSA conjugated peptides CKTDAELVKKKDE (amino acid 68–80) and GDAPPAKAEAKKDEK (amino acid 575–587) marked on Supplementary Fig. 1D. IF secondary: Alexa-488, -546, -647-coupled goat anti-mouse, goat anti-rabbit or goat anti-guinea pig (Life Technologies, 1:500). Western analysis: rabbit anti-CENP-A (Active Motif, #39713, 1:1000), mouse anti-ATPsyn-α (Abcam #ab14748, 1:1000), mouse anti-ATPsyn-β (Abcam #14730, 1:1000), rat anti-GST (Chromotek 6G9, 1:1000), mouse anti-red (Chromotek 6G6, 1:1000), mouse anti-poly-his (Sigma Aldrich #H1029, 1:1000), mouse anti-tubulin (Sigma Aldrich #T5168, 1:10,000) and rabbit anti-histone 3 (Millipore 17–10,254; 1:50,000); guinea pig anti-ATPsyn-βlike (1:1000). Uncropped scans of blots are provided in Supplementary Figure 5.