Anti acc

The total protein or nuclear protein from liver homogenates or cells was extracted using commercial kits (Keygen, Nanjing, China). Sample proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis in a Bio-Rad Mini protean apparatus (Bio-Rad, Hercules, CA, USA) and transferred to a PVDF membrane (Millipore, Billerica, MA, USA). The membranes were then blocked and incubated with primary antibodies (anti-SREBP-1, anti-ACC, anti-FAS, anti-CPT1A, anti-HADHB, anti-CROT, Santa Cruz Biotechnology, Dallas, Texas, USA) followed by incubation with a horseradish peroxidase-labeled secondary antibody (Santa Cruz Biotechnology, Dallas, Texas, USA). Densitometric analysis was performed directly from the blotted membrane using the Fusion FX5 imaging system (Vilber Lourmat, Marne, France).

The cytoplasmic proteins of liver tissues and hepatocytes were extracted according to the instructions of the cytoplasmic protein extraction kit (Beyotime, Beijing, China). A total of 30–60 μg cytoplasmic protein was resolved on an 8%–12% precast gel using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (Invitrogen, NY, USA) and transferred to polyvinylidene fluoride membranes (Bio-Rad, CA, USA). The membranes were then incubated with anti-p-AMPK, anti-AMPK, anti-G6Pase, anti-PEPCK, anti-ACC, anti-p-ACC, anti-FAS, anti-CPT-1a, anti-SHP1, anti-SREBP-1c (Santa Cruz, CA, USA), and anti-Glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Cell Signaling, MA, USA). Then, these membranes were washed with PBS and incubated with anti-mouse or anti-rabbit secondary antibody (Beyotime) for 1 h at room temperature. Finally, the immune complexes were developed using an enhanced chemiluminescence western blotting substrate, and protein expression levels were quantified using ImageJ software.