2.8. Western Blot Analysis for Lipid Metabolic Proteins in the Hepatic Tissue

Western blot analysis evaluated five hepatic proteins related to fatty acid metabolism as follows: mature sterol regulatory element-binding protein (mSREBP)-1c, fatty acid synthase (FAS), peroxisome proliferator-activated receptor (PPAR)-α, total AMP-activated protein kinase (AMPK)-α, and phosphorylated-AMPK (pAMPK)-α.

Regarding TG synthesis, western blot analysis was conducted to evaluate three hepatic proteins as follows: glycerol-3-phosphate acyltransferase (GPAM), diglyceride acyltransferase (DGAT)1, and DGAT2.

The proteins were separated by 10% polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes. After blocking in 3% BSA, the membranes were probed with primary antibodies (SREBP-1c, PPAR-α, GPAM, DGAT1, and DGAT2 (Abcam, UK); FAS, AMPK-α, and pAMPK-α (Cell Signaling Technology, Danvers, MA, CA, USA); β-actin from (Thermo Fisher Scientific, Waltham, MA, USA)) overnight at 4 °C. The membranes were washed and incubated with an anti-rabbit IgG-HRP (GeneTex, Irvine, CA, USA) for 2 h. Proteins were imaged using an advanced enhanced chemiluminescence (ECL) advanced kit (Thermo Fisher Scientific, CA, USA). Protein expression was observed using the FUSION Solo System (Vilber Lourmat, Marne-la-Vallée, France), and semi-quantified using ImageJ (NIH).