Ecl western blotting detection kit

Cells were lysed in ice‐cold RIPA buffer (Beyotime Biotechnology) and supplemented with phenylmethanesulfonyl fluoride (Beyotime Biotechnology). Protein concentrations were determined using the BCA protein assay kit (Beyotime Biotechnology). Total proteins were resolved by sodium dodecylsulfate–polyacrylamide gel electrophoresis and transferred to PVDF membranes (Millipore). After blocking, the membranes were incubated with a primary antibody and detected with a horseradish peroxidase‐conjugated secondary antibody. Proteins were visualized using ECL Western blotting detection kit (Advansta). Immunoreactive bands were quantified using Image Quant TL software. The following antibodies were used: anti‐PLPPR4 (Jackson, sc‐377263, 1:1000), anti‐phospho‐p44/42 MAPK (ERK1/2) (Cell Signaling Technology, 4370 s, 1:1000), anti‐p44/42 MAPK (ERK1/2) (Cell Signaling Technology, 4659, 1:1000), anti‐phospho‐mTOR (Cell Signaling Technology, 2971S, 1:1000), anti‐mTOR (Cell Signaling Technology, 2983S, 1:1000) and anti‐GAPDH (Cell Signaling Technology, 5174S, 1:1000). All antibodies were diluted in PBS containing 5% skimmed milk and 0.1% Tween‐20.

Adult males (7–10 days old) were frozen in liquid nitrogen (30 flies of each genotype) at the beginning of the day (1 h after lights on). Heads were cut off on ice and manually crushed in liquid nitrogen using a handheld homogenizer (LLG). Then, they were homogenized using an ultrasonic homogenizer (Dr. Hielscher) in 2× Laemmli Buffer with protease inhibitors (Boehringer). Samples were gently shaken, frozen at −20°C, thawed and centrifuged at 13,200 rpm for 1 h at 4°C. The supernatant was denatured at 80°C for 5 min. The protein concentration was measured using a Quant-iT Protein Assay Kit and a Qubit fluorometer (Invitrogen). Electrophoresis was performed using a NuPAGE SDS-PAGE Gel System. A 4–12% Bis-Tris gel (Invitrogen) was loaded with 10 μg of protein. The proteins were blotted by electrotransfer onto a PVDF membrane (Invitrogen) followed by blocking with 5% dry milk in PBS with 0.1% Tween-20 for 1 h at room temperature. Membranes were incubated with rabbit anti-DmMANF (1:5000) and anti-α tubulin (1:20000, Abcam 4074) antibodies as a loading control. For secondary antibody, we used goat anti-rabbit HRP (1:10000, Abcam 6721). The ECL Western blotting detection kit (Advansta) was applied for immunodetection. Densitometric quantification was performed using ImageJ software. The Western blot analysis was repeated in four independent experiments.