Foxo1

Total proteins of liver tissues were extracted using RIPA lysis buffer (Leagene Biotechnology, China). Protein concentrations were measured using bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, United States). Samples were analyzed by western blotting as previously described (29 (link)). Samples were loaded onto 8 or 10% SDS polyacrylamide gels (Beyotime Biotechnology, China), and then transferred to PVDF membranes (IPVH00010, Millipore, Germany) and immunoblotted using standard techniques Membranes were incubated with freshly mixed ECL solution (Biosharp, China) and visualized with an Automated Imaging System (Tanon, China). Antibodies used for western blotting were β-actin (Cell Signaling Technology, United States), PEPCK (Cell Signaling Technology, United States), PGC-1α (Cell Signaling Technology, United States), Akt (Cell Signaling Technology, United States), p-Akt (Cell Signaling Technology, United States), PKAα (Cell Signaling Technology, United States), p-PKA (Cell Signaling Technology, United States), GS (Abcam, United Kingdom), p-GS (Abcam, United Kingdom), PK (Abcam, United Kingdom), p-PK (Abcam, United Kingdom), AMPK (Abcam, United Kingdom), p-AMPK (Abcam, United Kingdom), FOXO1 (Bioworld, China), p-FOXO1 (Bioworld, China) and GPa (Bioworld, China).

Proteins were extracted from C18-4 cells which were treated with various doses of Res for 24 h. Cells were collected and lysed with lysis buffer, and then the protein concentration was detected using the BCA Protein Quantification Kit (Vazyme, Piscataway, NJ, USA). After heat denaturation in 5% SDS–PAGE sample loading buffer, the protein samples were resolved by SDS-PAGE and transferred to a PVDF membrane [55 (link)–56 (link)]. The samples were probed with β-ACTIN (1:1000; Sino Biological Inc., Shanghai, China), GAPDH (1:1000; Sino Biological Inc.), p53 (1:1000; Bioss, Beijing, China), Ac-p53 (1:1000; Cell Signaling Tecnology, Inc., USA), FOXO1 (1:500; Bioworld, Nnjing, China), Ac-FOXO1 (1:500; Cell Signaling Tecnology), BCL2 (1:500; Bioss), P38 (1:500; Sangon Biotech, Shanghai, China), SIRT1 (1:1000; Bioworld) as previously described [24 (link)]. The secondary antibody was horseradish peroxidase-conjugated anti-rabbit/mouse IgG (1:1,000; Boster, Wuhan, China). Protein blots were probed with the indicated primary antibodies and appropriate secondary antibodies and protein bands were visualized using the Thermo Scientific Pierce ECL western blotting substrate, and the results were analyzed using a Tanon-410 automatic gel imaging system.