Western Blotting Analysis of Insect Proteins

For Western blotting, equal amounts of protein extracts from whole insects were separated by SDS-PAGE and transferred onto PVDF. The membranes were blocked, incubated with the corresponding primary antibody. The primary antibodies for western blots were diluted with 5% skim milk in 0.1% Tween/TBS for anti-TcAK1 (1:1,000), anti-TcAK2 (1:2,000), anti-TcFOXO (1:1,000), anti-TcAMPKα (1:1,000), anti-α-tubulin (1:5,000) (Proteintech Group, Inc., Chicago, IL, USA), anti-Histone H3 (1:5,000) (Beyotime, Shanghai, China), Mouse Anti-AMPK alpha 1 + AMPK alpha 2 antibody (1:1,000), Recombinant Anti-GST antibody (1:5,000) (Abcam, Cambridge, MA, USA). The specificity of the TcAK1, TcAK2, TcFOXO and TcAMPKα antibodies was verified in our previous studies [29 (link), 58 (link), 59 (link)] The membranes were washed and incubated with the corresponding secondary antibody such as Goat anti-mouse IgG/HRP (1:10,000) (Solarbio, Beijing, China) and Goat anti-rabbit IgG/HRP antibody (1:10,000) (Solarbio, Beijing, China). The blot signals were detected using Tanon High-sign ECL Western Blotting kit (Tanon, Shanghai, China) with a ChemiDoc Touch Imaging System (Bio-Rad, Hercules, CA, USA) as described previously [29 (link)].