Mouse anti atp5a

For WB analysis, proteins were separated via SDS-page on a 12% SDS-polyacrylamide gel at 120V. A wet-transfer into a PVDF membrane was performed at 30 V over-night and membranes were blocked with 5% milk in TBS-T for 1 h at RT. Primary antibodies were diluted in 1% milk in TBST and incubated at 4°C over-night. After 3 washes, the secondary antibody, diluted in TBST, was incubated for 1 h at RT. After another 3 washes, specific proteins/bands were visualized with the Odyssey infrared imaging system (LI-COR). The following primary and secondary antibodies were used for WB analysis at the indicated concentrations: rabbit anti-HA (Abcam, 1:1000), rabbit anti-GST Z5 (Santa-Cruz, 1:1000), rabbit anti-CoxIV (Abcam, 1:1000), mouse anti-ATP5a (Abcam, 1:1000), rabbit anti-ADPR (N/A, 1:500), rabbit anti-ADPR (CST, 1:5000), IRDye 800CW goat anti-rabbit IgG (1:15,000, LI-COR, P/N 925-32211), and IRDye 680RD Goat anti-Mouse IgG (1:15,000, LI-COR, P/N 925-68070). For dot blot analysis, auto-modified proteins or isolated PAR chains were vacuum-blotted onto a nitrocellulose membrane, that was further blocked in milk and stained with antibodies as described above.

S2 cells were homogenized in a RIPA lysis buffer [5 mmol/L Tris–HCl, pH 8.0, 150 mmol/L NaCl, 1% (v/v) IGRPA CA-630, 0.5% (w/v) sodium deoxycholate and protease inhibitor cocktail (Roche)] and centrifuged at 12,000×g for 10 min at 4 °C to pellet nuclei and cell debris. The supernatant was collected and the protein concentration was assayed using a BCA protein assay kit (Beyotime). 20 μg of the protein samples were subjected to 12% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF (polyvinylidene fluoride) membranes. The blotted membranes were immunoblotted with primary antibodies: rabbit anti-Prominin-like (1:2000), mouse anti-Tubulin (1:1000, DHSB E7), mouse anti-VDAC (1:1000, Abcam), mouse anti-Cytochrome C antibody (1:1000, Abcam 13575), mouse anti-ATP5a (1:1000, Abcam), mouse anti-COX IV (1:1000, Abcam), Rabbit anti-CG9172 (ND20) (1:500, Abgent), rabbit anti-HA (1:1000, Abcam), mouse anti-Flag (1:1000, Cell Signaling), rabbit anti-LAMP-1 (1:1000, Abcam). After secondary anti-rabbit horseradish peroxidase antibody labelling (Pierce; 1:5000), ChemiLucent™ ECL detection reagents (Millipore) was used to detect proteins and images were taken using the Chemiluminescence Imaging System (Clinx Science Instruments) as previously reported [34 (link)].

Western blot analysis was performed as previously described [36 (link)]. Primary antibodies were diluted in TBS/0.1% Tween 20 (TBS-T) and incubated overnight at 4°C and were Drosophila anti-TFAM (Abcam, 1/500), mouse anti-ATP5A (Abcam, 1/5000), mouse anti-MTCO1 (Abcam, 1/1000) and rabbit anti-Actin (Cell Signaling, 1/4000). After three ten minute washes in TBS-T, the membranes were incubated for 90 minutes with fluorescently labelled secondary antibodies (anti-mouse IRdye 680 and anti-rabbit IRdye 800, LI-COR, both at 1/5000) diluted in TBS-T, then washed three times for ten minutes in TBS-T. The membranes were then scanned and analysed using an Odyssey infrared scanner (LI-COR). Odyssey infrared imaging systems application software version 3.0.25 was used to quantify the intensity of the bands on the blots. Normalised expression level was calculated by determining the band intensity relative to Actin.

RNA extraction and QRTPCR to measure AOX transcript levels using RpL32 RNA as an internal normalization standard were as previously described10 (link), using RNA from 2 day-old adult male and female flies. Protein extraction from 2 day-old Drosophila adults and Western blots were conducted essentially as by Fernandez-Ayala et al.10 (link), with the following modifications: for females, 1% SDS was used for lysis instead of 1.5% Triton X-100, flies were processed in batches of 30 (females) or 40 (males), SDS-PAGE used Any kD™ Criterion™ TGX™ 18-well gels (Bio-Rad), Prestained Protein Ladder (Thermo-Scientific) and ProSieve^TM EX Running and Transfer Buffers (Lonza), and membranes were treated in PBS-Tween® instead of TBS. Primary antibodies used were customized rabbit anti-AOX10 (link) (21^st Centrury Biochemicals, 1:10,000), rabbit anti-α-actininin C-20-R (Santa Cruz Biotechnology, 1:5,000) and mouse anti-ATP5A (Abcam, 1:50,000). Secondary antibodies were Peroxidase Goat Anti-rabbit IgG and Horse Anti-mouse IgG (both from Vector Laboratories, 1:10,000). Post-nuclear extracts (PN) from HEK293T cells were prepared according to Cannino et al.21 (link). Protein concentrations were measured using the Bradford assay.

For brain immunostaining, flies were fixed in 3.7% formaldehyde in phosphate buffered saline (PBS) for 20 min. After fixation, brains were dissected and fixed again for 5 min. Samples were then rinsed 3 times for 10 min with PBST and blocked in 3% BSA in PBST (PBST-BSA) for 1 hour. Primary antibodies were diluted in PBST-BSA and incubated overnight at 4°C. Primary antibodies used were: mouse-anti-ATP5a 1:250 (15H4C4, abcam); mouse anti-actin 1:50 (JLA20, DSHB), mouse-anti-FK2 1:250 (BML-PW8810–0500, ENZO); rabbit-anti-atg8a 1:250 (Rana et al., 2017). mouse-anti-dsDNA 1:250 (ab27156, abcam). Samples were then rinsed 3 times in PBST for 10 min. and incubated with the secondary antibodies and/or stained at room temperature for 3 hours. Secondary antibodies and stains used were: anti-rabbit or anti-mouse AlexaFluor-488 1:500 (A-11001 or A-11008, Thermo Fisher Scientific); anti-rabbit or anti-mouse AlexaFluor-568 1:500 (A-11031 or A-11036, Thermo Fisher Scientific); To-Pro-3 DNA 1:500 (T3605, Thermo Fisher Scientific; phalloidin AlexaFlour-568 1:250 (A12380, Thermo Fisher Scientific). Finally, samples were rinsed 3 times with PBST for 10 min and mounted in Vectashield Mounting Medium (Vector Lab). Images were taken using Zeiss LSM780 or LSM880 confocal microscope and analyzed with ImageJ software to measure intensity, area, count, and/or sizes of stained structures.

Heads from adult flies were collected and homogenized in lysis buffer (10 mM Tris–HCl, pH 7.4; 150 mM NaCl; 5 mM EDTA; 5 mM EGTA; 10% glycerol; 50 mM NaF; 1 mM Na₃VO_4,; 5 mM NaPPi; 5 mM DTT; 4 M urea with protease inhibitor cocktail) at 4 °C. Protein extracts were centrifuged at 3,000 g for 3 min at 4 °C, and the supernatants were stored at -20 °C. Lambda phosphatase (New England Biolabs) was added to the thawed protein extracts and incubated at 30 °C for 30 min. Western blotting was performed following the standard procedure^{45 (link)}. The primary antibodies were used with the following dilutions: rabbit anti-human pan tau (Dako, 1:20,000), mouse anti-tau-C3 (Invitrogen, 1:10,000), mouse anti-α-tubulin (GeneTex, 1:5,000), mouse anti-β-tubulin (Developmental Studies Hybridoma Bank, 1:5,000), mouse anti-AT8 (Thermo, 1:500), mouse anti-AT100 (Thermo, 1:500), mouse anti-ATP5a (Abcam, 1:100,000), rabbit-histone H3 (Abcam, 1:5,000), and mouse anti-syntaxin (Developmental Studies Hybridoma Bank, 1:5,000). Secondary antibodies conjugated with HRP (Jackson ImmunoResearch Laboratories) were used in 1:10,000 dilutions. All loading controls were prepared by stripping off the reagents from the original membrane and then re-immunoblotting following the standard procedures. Semiquantitative analysis of band density was performed in ImageJ.

Detection of sEV proteins was performed by immunoblotting as previously described (van Balkom et al., 2015 (link)). For immunoblotting, sEV and cell samples were diluted 1:1 in Exosome Sample Buffer (5% SDS, 9 M urea, 10 mM EDTA, 120 mM Tris‐HCl, pH 6.8, 2.5% beta‐mercaptoethanol) and heated (95°C, 5 min). For cell samples, cells were scraped from the culture surface, resuspended in lysis buffer (1% SDS and 0.1% Triton X‐100 in PBS with protease inhibitors [cOmplete Mini, EDTA free, Roche] and incubated on ice for 30 min. Genomic DNA was sheared through a 27G needle four times. SDS‐PAGE, protein transfer and blocking were performed as described (van Balkom et al., 2015 (link)). Subsequently, membranes were incubated in either of the following antibodies: rabbit‐anti‐GAPDH (Cell Signaling), rabbit‐anti‐Flotillin‐1 (Santa Cruz Biotechnology), goat‐anti‐Lamin A/C (Santa Cruz Biotechnology), mouse‐anti‐ATP5A (Abcam), or rabbit‐anti‐Tom20 (Santa Cruz Biotechnology; Table 1).
As secondary antibodies 1:2000 diluted affinity‐purified swine‐anti‐rabbit, rabbit‐anti‐mouse, or donkey‐anti‐goat coupled to horseradish peroxidase (Dako) were used. Antigen‐antibody reactions were visualised with enhanced chemiluminescence according to manufacturer's guidelines (Chemiluminescent Peoxidase Substrate, Sigma) and imaged using a GelDoc XR+ system (Bio‐Rad).