α atp5a

Immunostainings were carried out as described^{30 (link)} with the following modifications. Ovaries were dissected from 5–10 day old female flies in PBST (PBS with 0.3% Triton-100) and fixed in PBST with 4% formaldehyde for 20 min at room temperature (RT). Fixed ovaries were washed at least 3 times with PBST, permeabilized in PBS with 1% Triton X-100 for 1 h, and blocked in PBSTA (PBST with 1% bovine serum albumin) for 1 h. Samples were incubated with primary antibodies overnight at 4 °C in PBSTA. The dilutions of the primary antibodies were as follows: α-RpS5b (peptide antibody generated by Biomatik) 1:1000; α-RpS5a (peptide antibody was generated by Biomatik) 1:1000); α-αTubulin (Sigma) 1:5000; α-Orb (DSHB) 1:50; α-Dhc (DSHB) 1:50; α-cleaved Caspase 3 (Abcam), 1:200; α-ATP5A (Abcam) 1:1000; α-Aub 1:1000; α-Osk 1:500; α-Grk 1:500. Antisera were produced in the Lasko lab unless otherwise noted. Venus-tagged proteins were imaged directly under ultraviolet light.
Samples were washed and incubated in the dark with fluorescent secondary antibody (preadsorbed goat anti-rabbit Alexa Fluor555, or goat anti-mouse Alexa Fluor488, Molecular Probes, 1:500) in PBSTA for 90 min at RT. Samples were then dark washed and counterstained with DAPI, mounted in 1% DABCO (in 90% glycerol) anti-fade reagent, and examined under confocal microscopy (Zeiss LSM510).

Antibodies used in this work include: α-QIL1 (Sigma-Aldrich, St. Louis, MO; SAB1102836), α-MINOS1 (MIC10) (Aviva, San Diego, CA; ARP44801-P050), α-CHCHD3 (MIC19) (Aviva; ARP57040-P050), α-CHCHD6 (MIC25) (Proteintech, Rosemont, IL; 20639-1-AP), α-IMMT (MIC60) (Abcam, Cambridge, MA; ab110329), α-APOOL (MIC27) (Aviva ; OAAF03292), α-APOO (MIC26) (Novus Bio, Littleton, CO; NBP1-28870), α-ATP5A (Abcam; ab14748) and α-α-Tubulin (Cell Signaling Technologies, Danvers MA; #2125).

Western blots were performed using ExpressPlus PAGE Gel 4–12% or 4–20% (GenScript, Piscataway, NJ, USA). Proteins were transferred to PVDF membranes (MERCK-Millipore) using the Trans-Blot Turbo Transfer System (Bio-Rad, Hercules, CA, USA) following manufacturer’s instructions. Membranes were incubated with indicated antibodies and imaged with ImageQuant LAS4000. Band densiometry quantification was performed using ImageJ software. The following antibodies were used: anti-Actin (1:1000; Chemicon MAB1501, Tokyo, Japan), α-ATP5A (1:4000, Abcam ab14748, Cambridge, MA, USA), α-Cyclophilin D (1:500, Abcam ab110324), α-TOM20 (1:1000, Santa Cruz sc-11415, Dallas, TX, USA) and α-VDAC (1:1000, Abcam ab15895). Canonical secondary antibodies used were sheep anti-mouse or donkey anti-rabbit HRP (GE Healthcare, Chicago, IL, USA). Immunoreactivity was visualized with Immobilon Forte Western HRP substrate (Millipore, Burlington, MA, USA).