Ab110334

For each specimen, four thoraxes were dissected and homogenized by dounce tissue grinder in RIPA buffer containing protease inhibitor (cOmplete^TM, Roche). Cellular debris were removed by centrifugation at 4 °C, 14000× g for 20 min. The supernatants were collected and the protein concentrations were determined by Pierce protein assay (Pierce 660 nm Protein Assay Reagent, ThermoScientific). 0.6 μg/well of proteins were loaded for SDS-PAGE and western-blot analysis.
The primary antibodies and the dilutions were as follows: mouse anti-ATP5A (50000x, abcam ab14748), mouse anti-Cytochrome C (10000x, abcam ab13575), mouse anti-PDHA1 (1000x, abcam ab110334), rabbit anti-SOD2 (10000x, abcam ab13534) and rabbit anti-alpha Tubulin (10000x, abcam ab18251). The secondary antibodies and the dilutions were as follows: anti-mouse IgG-HRP (2000x, Invitrogen 62–6520), or anti-rabbit IgG-HRP (5000x, abcam ab97051). The signals were developed by chemiluminescent HRP substrate (Immobilon^TM Western, Millipore) and recorded by a digital multispectral imaging system (BioSpectrum, UVP).

HIF-1α antibody 1:1000 (abcam reference #ab51608), GLUT1 antibody 1:1000 (Merk Millipore reference #07-1401), PDK1 antibody 1:1000 (abcam reference #ab110025), HIF-1α OH-P402 1:1000 (abcam reference #ab72775), Beta-actin antibody 1:10000 (cell signaling, Cat. #4967), Pyruvate dehydrogenase E1-alpha subunit 1:2000 (abcam reference #ab ab110334) and Pyruvate dehydrogenase E1-alpha subunit (phospho S293) 1:1000 (abcam reference #ab177461) were used for western blot. GLUT1 and PDK1 were used for Immunohistochemistry assays.

The cells were lysed using lysis buffer (Biosharp) to obtain total protein. Next, loading buffer containing 5% 2‐mercaptoethanol was mixed with the lysate. The proteins were denatured at 100°C for 10 minutes before being separated on 8%‐12% sodium dodecyl sulfate‐polyacrylamide gels. After transfer to PVDF membranes (Millipore), the membranes were incubated for 2 hours at room temperature in TBST containing 5% skim milk. Afterward, membranes were incubated with the corresponding primary antibodies overnight at 4°C according to the manufacturer's instructions. After treatment with the appropriate horseradish peroxidase (HRP)‐conjugated secondary antibodies, the membranes were incubated with ECL western blotting reagents (Millipore). The following antibodies were used: SIRT3 (s4072; Sigma), cMYC (ab32072; Abcam), HIF1α (36169; CST, Danvers, MA, USA), PDK1 (5662; CST), p‐PDHA1 (ab92696; Abcam), GAPDH (ab128915; Abcam), Actin (A5441; Sigma), PDHA1 (ab110334; Abcam), GLUT1 (ab115730; Abcam), Anti‐mouse IgG (7076; CST), and Anti‐rabbit IgG (7074; CST).

Whole flies or dissected fly tissue were homogenised in 2x Laemmli loading sample buffer (100 mM Tris pH 6.8, 20% glycerol, 4% SDS; Bio-Rad) containing 50 mM DTT. Extracts were cleared by centrifugation and approximately 20–40 μg of protein extract was loaded per lane on a polyacrylamide gel. Proteins were separated and transferred to a nitrocellulose membrane. The following antibodies were used at the indicated dilutions: β-actin (Abcam, ab8227; 1:4000), Atg8 (a generous gift from Katja Köhler; 1:2000; [80 (link)], PDH (Abcam, Ab110334), NDUFS3 (Abcam, ab14711), GAPDH (GeneTex, GTX100118), cytochrome C (BioLegend, 612503, clone 7H8.2C12), SDHB (Abcam, ab14714), ATP5A (Abcam, ab14748), VDAC (Abcam, ab14734). The HRP-conjugated anti-rabbit secondary (Abcam, ab6721; 1:12000) was used. Blots were developed using the ECL detection system (GE, Amersham), and analysed using FIJI software (US National Institutes of Health). To quantify total protein, we used TGX stain-free gels from Bio-Rad (567–8123 or 567–8124) according to the manufacturer’s instructions. For hydroxychloroquine (HCQ) treatment, HCQ was dissolved in water to give a 10M stock. The stock was then added to standard SYA fly food to give a final concentration of 30mM. Flies were pretreated 24h with HCQ before western blot analysis.

Thin sections placed on nickel grids were blocked with 10% BSA in PBS for 20 min, and incubated with primary antibodies in incubation buffer (1% BSA in PBS) for 2 hr. Grids were subsequently washed with incubation buffer three times (10 min each). Secondary antibodies, goat anti-mouse IgG (EM.GMHL15, BB International) and protein A (EM.PAG15, BB International) conjugated to 15 nm gold particles, were used against the primary antibodies from mouse and rabbit respectively. Secondary antibodies at 20-fold dilution were applied and samples were incubated for 1 hr. After washing with PBS, the immune-complexes were fixed with 1% glutaraldehyde in PBS and washed three times with distilled water. The specimens were inspected by TEM operating at 120 kV (FEI Tecnai G2 TF20 Super TWIN).
The primary antibodies and applied dilution factors are listed as follows: mouse anti-dsDNA (500x, abcam ab27156), mouse anti-ATP5A (500x, abcam ab14748), mouse anti-Cytochrome C (8000x, abcam ab13575), mouse anti-PDHA1 (500x, abcam ab110334), mouse anti-Ubiquitin (1000x, abcam ab7254), mouse anti-DNA-RNA hybrid (500x, kerafast ENH001), and rabbit anti-SOD2 (500x, abcam ab13534).