Western Blot Analysis of Tissue Proteins

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).