Liver and Adipose Tissue Protein Expression

Western blot analysis was performed as previously described [18 (link)]. Briefly, frozen liver and adipose tissues from 3 chickens slaughtered per group were lysed using RIPA lysis buffer (IN-WB001, Invent, Plymouth, MA, USA) with 1 mM PMSF (P0100, Solarbio Technology Co., Beijing, China). The lysates were then diluted with Sample Loading Buffer (P0015L, Beyotime, Shanghai, China), boiled, and loaded onto an 8% SDS–PAGE. After electrophoresis, the proteins were transferred to a PVDF membrane and the membranes were routinely washed and blocked with 5% non-fat dry milk in TBST. Then, the following primary antibodies were used: anti-mTOR (T55306F, 1:1000 dilution), anti-phospho-AKT (Ser473) (T40067F, 1:2000 dilution), anti-AKT (T55561F, 1:2000 dilution), and anti-β-Tubulin (M20005F, 1:5000 dilution), all purchased from Abmart Shanghai Co., Ltd. (Shanghai, China). After applying the secondary antibodies, the membranes were developed using chemiluminescence (Abbkine, Waltham, MA, USA) and the signals were detected using a Bio-Rad Imaging System (Bio-Rad, Hercules, CA, USA).