Foxo3a

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).

After relevant treatment, cells were washed with cold PBS and lysed in RIPA buffer containing protease and phosphatase inhibitors. Equal amount of protein was subjected to sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis under reducing conditions, proteins transferred to nitrocellulose membrane and subjected to Western blot analysis with antibodies to FOXO3a, MDA5, TLR3, MAVS (Santa Cruz Biotechnology, Santa Cruz, CA), total and phospho-IRF3 (Cell Signaling), or β-actin (Sigma Aldrich, Saint Louis, MO). Specific bands were quantified by densitometry using NIH Image J, and the results expressed as fold change over β-actin or respective total protein. To determine MAVS aggregation, we performed semidenaturing detergent agarose gel electrophoresis (SDD-AGE) under non-reducing conditions as described previously using 1.5% agarose^{22 (link)} with some modifications. Briefly, mitochondrial crude extracts were resuspended in sample buffer (0.5 X TBE containing 10% glycerol, 2% SDS and 0.00025% bromophenol blue) and loaded on horizontal 1.5% agarose gel and the gel was run at 50 volts at 4 °C overnight. The separated proteins were passively transferred to immobilon overnight and subjected to Western blot analysis with an antibody to MAVS.