Dw101 01

Fat bodies of 40 third instar larvae were extracted with PBST containing protease inhibitors (APExBIO, K1007) and phosphatase inhibitor cocktail (Beyotine, P1081), and the protein concentration was measured by the BCA kit (Thermo Scientific, 23227). Equal amounts of total protein were subjected to western blotting [42 (link)]. Fat body samples were separated by SDS-PAGE in 10% gels and transferred to PVDF membranes (Millipore). The primary antibodies used were rabbit anti-pJNK (Millipore, 07–175, 1:2000), anti-JNK (zen-bio, AF6318, 1:1000), anti–Myc (DSHB, P4C4-B10, 1:100) and anti-GADPH (Servicebio, GB11002, 1:2000). Secondary peroxidase–conjugated antibodies used were goat anti-mouse (BOSTER, BA1054, 1:5000) and goat anti-rabbit (BOSTER, BA1050, 1:5000). Signals were developed with an ECL detection kit (TransGen, DW101-01). The images were acquired with a gel documentation system (ProteinSimple, FluorChem E). The signals were measured with ImageJ software. All the experiments were repeated at least three times.

Proteins were lysed and extracted by RIPA buffer (89900, Thermo Scientific, United States). Subsequently, proteins were separated by SDS-PAGE and transferred to PVDF membranes. The primary antibodies used for immunoblotting were PI3K (1:4,000, YM3408, Immunoway, China), AKT (1:4,000, 4691, CST, United States), p-AKT (S473, 1:4,000, YP0006, Immunoway, China), P53 (1:4,000, Ab26, Abcam, China), P21 (1:2,000, ab109199, Abcam, China), P16^INK4a (1:5,000, ab108349, Abcam, China), GAPDH (1:5,000, YM3029, Immunoway, China), and β-Actin (1:5,000, YM3028, Immunoway, China). The immunoreactive bands were visualized via electrochemiluminescence (ECL, DW101-01, TransGen Biotech, China) method and analyzed by ImageJ software (ver. 1.44, National Institutes of Health, United States).