Ir dye 680 or 800cw labeled

Brain tissues stored at −80 °C were solubilized in a lysis buffer, and lysates were separated on 10% SDS-PAGE gel. Proteins were then transferred onto a PVDF membrane. Phospho-cAMP-response element-binding protein (p-CREB), Phospho-protein kinase A (p-PKA), Phospho- extracellular regulated protein kinases (p-ERK1/2), Phospho-protein kinase B (p-AKT), Phospho-Ca²⁺/calmodulin-dependent protein kinase II (p-CaMKII), Phospho-S6 ribosomal protein (p-S6), β-tubulin and β-actin were detected by immunoblotting with the antibody (1:1000–1:4000). Blots were probed by goat anti-rabbit or goat anti-mouse second antibody with IR-Dye 680 or 800 cw labeled (1:2000–1:4000, Licor, Lincoln, NE, USA) at room temperature for 1 h. The membranes were then visualized using a LiCor Odyssey scanner (Licor, Lincoln, NE, USA) and quantified with ImageJ 1.44 software (National Institute of Health, Bethesda, MD). The phosphorylation levels of the cAMP-response element-binding protein (CREB), the cAMP-dependent protein kinase (PKA), the extracellular regulated protein kinases (ERK1/2), protein kinase B (AKT), Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) and S6 ribosomal protein (S6) were normalized according to the loading of proteins by expressing the data as a ratio over β-actin or β-tubulin.

The whole fish (13 dph) and liver tissues (83 dph) stored at − 80 °C were solubilized in RIPA lysis buffer. The protein content was determined using BCA protein assay kit (Yeasen, China). The proteins were separated on 10% SDS-PAGE gel, and then transferred onto PVDF membrane. Anti-phospho ribosomal protein S6 kinase 1 (S6K1) (Thr389), anti-S6K1, anti-phospho ribosomal protein S6 (S6) (Ser235/236), anti-S6 and anti-phospho Grb10 (Ser476) were purchased from Cell Signalling Technology (USA), anti-β-actin antibody from Bioss (China), anti-β-tubulin antibody from Zoman Biotechnology (China). Blots were probed by goat anti-rabbit and goat anti-mouse second antibody with IR-Dye 680 or 800cw labeled (Licor, USA) at room temperature for 1 h. The membranes were then visualized using a LiCor Odyssey scanner (Licor, USA) and quantified with ImageJ 1.44 software (National Institute of Health, MD). The phosphorylation level of S6 and S6K1 were normalized according to the loading of proteins by expressing the data as a ratio of phospho-S6 and phospho-S6K1 over S6 and S6K1, respectively. Besides, the phosphorylation level of Grb10 were normalized according to the loading of proteins by expressing the data as a ratio of phospho-Grb10 over β-actin.