*2.7. Western Blotting Analysis*

Western blotting was used to detect the protein expression levels of *Es*FOXO-like with the commercial FOXO1 antibody (ab52857, Abcam, Cambridge, MA, USA). The proteins were extracted from the hepatopancreas using the Protein Extraction Kit (Biyotime, Beijing, China) according to the protocol. Samples were separated by 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes. The membranes were blocked with 3% Bovine Serum Albumin (BSA) (Sangon Biotech, Shanghai, China) in TBST (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.2% Tween-20) at 4 ◦C overnight. Then, the membranes were incubated with 1/3000 diluted FOXO1A antibody or beta-tubulin rabbit antibody (Beyotime Biotechnology, Beijing, China) in TBST with 3% BSA for 2 h at room temperature, and this was followed with washing in TBST three times. Next, the membranes were incubated with HRP-labeled goat anti-rabbit IgG (Beyotime Biotechnology, Beijing, China) at a ratio of 1:1000 (diluted in 3% BSA) for 1 h at room temperature. After three washes, the membranes were incubated in the western blot chemiluminescence HRP substrate (Beyotime Biotechnology, Beijing, China) in the dark for 2 min, and the protein bands were visualized using the chemiluminescent imaging system (Amersham Imager 600, USA). The relative protein expression levels of *Es*FOXOlike were analyzed by ImageJ software (http://rsb.info.nih.gov/ij/) (accessed on 17 March 2023). All bands were digitalized by using the ImageJ software.
