Mouse anti actin

For ovary lysates, ovaries were dissected and placed immediately on dry ice. Cell and ovary lysates were extracted using radioimmunoprecipitation assay lysis buffer (Santa Cruz Biotechnology, Inc.) plus complete protease inhibitors (Roche). Protein was quantitated using the Bicinchoninic Acid Protein Assay (Thermo Fisher Scientific). Proteins were separated on a 4–12% NuPAGE Bis-Tris gel (Invitrogen) and transferred to polyvinylidene fluoride membranes. Membranes were blocked in milk or Odyssey blocking buffer and incubated in primary antibodies overnight at 4°C. Primary antibodies included rabbit anti-Ref(2)P (1:10,000), mouse anti-actin (1:500; JLA20; Developmental Studies Hybridoma Bank), mouse anti-Tubulin (1:1,000; E7), mouse anti–ATPsyn-α (1:1,000), mouse anti-Porin (1:1,000; MitoSciences), mouse anti-ANT (1:500; MitoSciences), rabbit anti–Dcp-1 (1:500; Laundrie et al., 2003 (link)), guinea pig anti–Dcp-1 (1:500; Tenev et al., 2005 (link)), rabbit anti-Pink1 (1:500; Abcam), and rabbit anti-Atg8a (1:1,000; Barth et al., 2011 (link)). Membranes were incubated with HRP-conjugated secondary antibodies or infrared-labeled secondary antibodies and were detected using the ECL Enhanced Western Blotting System (GE Healthcare) or the Odyssey System (LI-COR Biosciences). Densitometry was performed using ImageQuant 5.1 software (GE Healthcare).

For immunoblotting of heads extract, 40 heads of male flies were collected and frozen at indicated time. Heads were homogenized with T300 buffer, and protein samples were loaded onto acrylamide gels. Proteins were transferred on nitrocellulose membranes (GE health), and the membranes incubated with rat anti-TANGO10 (Fig. 1 and SI Appendix, Fig. S4), mouse anti-TANGO10 (SI Appendix, Fig. S5), mouse anti-synapsin (Developmental Studies Hybridoma Bank 3C11), rat anti-GE-1 (73 (link)), mouse anti-CUL3 (BD Biosciences 611848), mouse anti-V5 (ThermoFisher), rat anti-HA (Sigma Aldrich), rabbit anti-PER (74 (link)), guinea pig anti-TIM (10 (link)), mouse anti-actin (Developmental Studies Hybridoma Bank, JLA20), and anti-FLAG (Sigma-Aldrich) antibodies. The blots were detected using enhanced chemiluminescence prime (GE Healthcare). Quantifications were performed using Image J from two separate experiments (SI Appendix, Fig. S5).