Rabbit anti cox 4

Protein samples were separated by 12% or 16% SDS polyacrylamide (SDS-PAGE) gel electrophoresis followed by transferring to PVDF membranes (GE Healthcare). Membranes were incubated in blocking solution containing 5% skimmed milk in TBST (10 mM Tris–HCl pH 7.6, 150 mM NaCl and 0.1% Tween 20) for 1 h prior to incubation with primary antibodies for either 1 h at room temperature or overnight at 4 °C. After washing with TBST three times, membranes were incubated with appropriate HRP-conjugated secondary antibodies (1:5000, GE Healthcare). Blots were then developed with the ECL kit (GE Healthcare). Primary antibodies were: rabbit anti-TOM20 (1:200, Santa Cruz), rabbit anti-VDAC (1:1000, Cell Signaling), rabbit anti-cytochrome-c (1:1000, Cell Signaling), rabbit anti-COX IV (1:2000, Abcam), rabbit anti-HSP-60 (1:1000, Cell Signaling), mouse MitoProfile total OXPHOS rodent WB antibody cocktail (1:250, Abcam), mouse anti-c-Myc (1:5000, Sigma-Aldrich), rabbit anti-GAPDH (1:5000, Sigma-Aldrich; or 1:1000, Cambridge Bioscience), mouse anti-Mfn1 (1:250, Abcam), mouse anti-Mfn2 (1:250, Abcam), rabbit anti-Fis1(1:500, Santa Cruz), rabbit anti-Drp1 (1:1000, Cell Signaling), rabbit anti-phospho-Drp1 (Ser616) (1:1000, Cell Signaling), goat anti-OPA1 (1:500, Santa Cruz).

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.

To visualize hemocytes and GFP-based ROS signal, larvae were carefully washed in water using a fine paint-brush, dried on tissue paper and embedded on microscope slides in a drop of ice-cold 80% glycerol. The larvae were immobilized at -20°C for 24 h before live imaging using a Zeiss ApoTome.2 structured illumination microscope. For subcellular localization by immunocytochemistry, Drosophila S2 cells were transformed with pMT/V5-His A constructs using Fugene HD (Promega) according to manufacturer’s protocol. After induction with 500 μM Cu₂SO₄ for 48 h, cells were fixed and V5-tagged proteins and endogenous COXIV were detected as described previously [61 (link)], using mouse anti-V5 (Life Technologies, 1:1000) and rabbit anti-COXIV (Abcam, 1:300) as primary antibodies, respectively with AlexaFluor 568 goat anti-mouse IgG (H+L) and goat anti-rabbit AlexaFluor 488 IgG (H+L) (Life Technologies) as secondary antibodies (1:1000 in both cases). Samples were mounted in ProLong Gold Antifade Mountant with DAPI (ThermoFisher Scientific, P36931), according to manufacturer’s protocol.