Fgf21

Western blot analysis was performed with the same method used in a previous study [25 (link)]. Cytosolic and nuclear proteins were extracted from the liver, gastrocnemius tissue, and hippocampus with lysis buffers containing protease/phosphatase inhibitor, cytosol-extracting buffer. A total of 30 μg of protein for each group was used for western blot analysis. The extract was separated by 8~12% SDS-PAGE gel electrophoresis then transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). Then, it was incubated overnight with primary antibodies and probed with the respective secondary antibody reagent (Biorad, Hercules, CA, USA). The bands were adjusted by the band levels of α-tubulin (cytosol) or PCNA (nucleus) [25 (link)].
Antibody list: pAkt (sc-81434), Akt (sc-514032), GLUT4 (sc-53566), GLUT2 (sc-518022), FGF21 (sc-81946), KYN (sc-69890), PPARα (sc-398394), FGFR1 (sc-57132), CTSB (sc-365558), PGC1α (sc-517380), Foxo3a (sc-48348), Atroin-1 (sc166806), Murf-1 (sc-398608) (Santa Cruz Biotechnology, CA, USA, 1:200), IRS-1 (#2382), pIRS-1 (#2381), mTOR (#2972), pmTOR (#2971), LC3 (#2775), (#2535), AMPK (#2532) (cell signaling technology, MA, USA, 1:2000), FNDC5 (ab131390), BDNF (ab108319), β-klotho (ab127879) (Abcam, Cambridge, MA, USA, 1:10,000), α-tubulin (T5168, Signa Aldrich, MO, USA, 1:4000), PCNA (610665, Enzolife science, Farmingdale, NY, USA, 1:1000).

DHI was kindly provided by Jinan Buchang Pharmaceutical Co. Ltd (Shandong, China) and prepared as described21 (link). Rabbit anti-caspase-3 (CAS-3), fibronectin, G6Pase and FGF21 polyclonal antibodies were obtained from Santa Cruz Biotechnology (Dallas, Texas). Rabbit anti-VEGFA, PCK1, GCK, IRS1 and PPARγ polyclonal antibodies were purchased from Proteintech Group (Chicago, IL). Goat anti-AGEs, rabbit anti-AMPKα and phosphorylated AMPKα (pi-AMPKα) polyclonal antibodies were purchased from Novus Biologicals (Littleton, CO). Triglyceride (TG) assay kit was purchased from Wako Chemicals (Neuss, Germany). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO) except as indicated.

The dissected liver tissues were stored in liquid nitrogen until analyses (n = 6 per group). Otherwise, insulin (0 and 10 U/kg) was intraperitoneally administrated 15 min prior to tissue collection in certain rats (n = 6 per group). The tissues were homogenized in tissue-protein extraction reagent (T-PER, Pierce Biotechnology, Rockford, IL, USA). After sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) separation and transfer, the polyvinylidene fluoride (PVDF) membranes reacted with primary antibodies, horseradish peroxidase-labeled IgG, subsequently followed by Enhanced Chemiluminescence (ECL) detection reagents. The intensity of recognized protein was quantified through densitometry. The primary antibodies recognized included GLP-1R (1:1000, sc-390774), Klotho (1:500, sc-515939), Akt (1:2000, sc-8312), phospho-Akt (1:1000, sc-7985-R), ERK1/2 (1:2000, sc-514302), phospho-ERK1/2 (1:1000, sc-7383), PERK (1:1000, sc-9479), phospho-PERK (1:1000, sc-32577), FGF-21 (1:500, sc-16842), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:2000, sc-32233) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), FGFR1 (1:1000, #9740), phospho-FGFR1 (1:1000, #3471) (Cell Signaling, Danvers, MA, USA).