2.7. Western Blot Analysis

Liver tissues were prepared for Western blot as previously described [16,29,31]. Briefly, proteins were separated on a 10% SDS-PAGE gel and electroblotted onto nitrocellulose membranes. Nonspecific binding sites were blocked using defatted milk proteins and membranes were incubated overnight at 4 °C in blocking solution with the following primary antibodies: acetyl CoA carboxylase (ACC, 280 kDa, 1/1000) Cell Signaling, Danvers, MA, USA, AKT (60 kDa, 1/1000) Cell Signaling, fatty acid synthase (FAS, 273 kDa, 1/1000) Cell Signaling, phospho-p38 mitogen-activated protein kinases (p-p38 MAPK, 43 kDa, 1/1000) Cell Signaling, phospho stress-activated protein kinases/Jun amino-terminal kinases (p-SAPK/JNK, 46 kDa, 1/500) Cell signaling, SAPK/JNK (46 kDa, 1/1000) Cell Signaling; carnitine palmitoyl transferase 1A (CPT1A, 88 kDa, 1/1000) Cell Signaling, glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 1/1000, 37 kDa) Invitrogen, superoxide dismutase 2 (SOD2, 21 kDa, 1/3000) Invitrogen; glutathione peroxidase 1 (GPx1, 26 kDa, 1/1000) Novus Biologicals, Centennial, CO, USA, interlukin-6 (IL-6, 24 kDa, 1/1000) Novus Biologicals; glucose-6-phosphatase (G-6-Pase, 1/1000, 58 kDa) Abcam, Cambridge, UK, nuclear factor erythroid-2-related factor 2 (NRF2, 110 kDa, 1/1000) Abcam, peroxisome proliferator activated receptor coactivator 1a (PGC-1α, 130 kDa, 1/1000) Abcam, phosphoenolpyruvate carboxykinase (PEPCK, 62 kDa, 1/1000) Santa Cruz, Dallas, TX, USA; peroxisome proliferator-activated receptor alpha (PPARα, 57 kDa, 1/1000) Cayman Chemical, Ann Arbor, MI, USA; phospho-AKT^Ser473 (p-AKT, 60 kDa, 1/1000) ThermoFisher Scientific, p38 MAPK (43 kDa, 1/1000) ThermoFisher Scientific; sterol regulatory element-binding protein 1 (SREBP1c, 60 kDa,1/1000) Abcam; toll like receptor 4 (TLR4, 79 kDa, 1/1000) Abcam; TNF-alpha (TNF-α, 26 kDa, 1/1000) ThermoFisher Scientific; phospho-ACC (p-ACC, 257 kDa, 1/1000) Millipore; β-actin (43 kDa, 1/250,000) Sigma-Aldrich, Saint Louis, MO, USA. The relative amount of primary antibody was detected with a species-specific horseradish peroxidase-conjugated secondary antibody using a ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA, USA). All data are expressed as the ratio of target protein to β-actin or GAPDH (used as housekeeping genes).