Chemiluminescent peroxidase substrate 3 kit

For the phosphorylated histone Western blot, landing board bees were fed sucrose solution containing 2.5 or 25 µg/mL bleomycin for 48 hours or sucrose solution alone. Total lysates from five pooled midguts were generated by pestle-mediated homogenization of remaining midgut tissues in RIPA buffer containing protease inhibitors (Sigma, St. Louis, MO, USA) and phosphatase inhibitors (Halt Phosphatase Inhibitor, Thermo Scientific, Waltham, MA, USA). Following incubation on ice to allow for cellular lysis, lysates were centrifugated to remove cellular debris. For Western blot analysis, aliquots of total lysates containing 50 µg of total protein were fractionated on an SDS-polyacrylamide gel and electroblotted onto polyvinylidene difluoride membrane. Antibody incubation and chemiluminescence detection (using the Chemiluminescent Peroxidase Substrate-3 kit) were performed according to the manufacturer’s instructions (Sigma, St. Louis, MO, USA). The antibodies used include those directed to GAPDH (HRP-60004, Proteintech, Rosemont, IL, USA) directly conjugated to HRP and pHistone H2AvD (Ser137) (600-401-914, Rockland Immunochemicals Pottstown, PA, USA) followed by use of Goat anti-rabbit secondary conjugated to HRP. Western blot visualization was performed using a Bio-Rad ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA, USA).

Cells were lyzed in radioimmunoprecipitation assay (RIPA) buffer (20 mM Tris HCl pH 7.4, 150 mM NaCl, 4 mM EDTA, 1% Triton X-100, and 0.2% SDS) supplemented with phosphatase inhibitors (1 mM Na₃VO₄, 1 mM NaF) and 1 mM phenylmethylsulfonylfluoride (PMSF), purchased from Sigma-Aldrich, and protease inhibitor cocktail (Roche Applied Science) (Penzberg, Upper Bavaria, Germany). Protein lysates were fractionated on SDS-PAGE (10 or 15%), and transferred to a nitrocellulose membrane (Whatman) (GE Healthcare, Little Chalfont, England). Western-blotting was performed using LC3-B (Sigma-Aldrich #L7543), ATG7 (Cell Signaling Technology #2631), Beclin-1 (Cell Signaling Technology #3738), ALK (D5F3 XP, Cell Signaling Technology #3633), ULK1 (Cell Signaling Technology #4773), GAPDH (Millipore MAB374) and β-actin (Santa Cruz #7210) antibodies. Proteins were visualized using the Chemiluminescent Peroxidase Substrate-3 Kit (Sigma-Aldrich) or the ECL™ Prime Western Blotting Detection Reagent (Amersham Biosciences) (Buckingshire, UK).