P acc

Differentiated L6 myoblasts treated with OFS were lysed, and equal amounts of protein were subjected to SDS-PAGE and immunoblotted with antibodies specific for AMPK, p-AMPK (Thr 172), ACC, p-ACC, JNK, p-JNK, ERK, p-ERK, p38, p-p38, GLUT4 and β-actin (Cell Signaling Technology, Beverly, MA, USA). Immunoreactive bands were visualized using chemiluminescent imaging system (Fusion SL2, Vilber Lourmat, Marne-la-Vallée Cedex, France), and analyzed by Bio1d software (Vilber Lourmat, Marne-la-Vallée Cedex, France).

HepG2 cells were harvested and boiled with a sample buffer (1 × DB). Proteins were separated by SDS-PAGE and transferred to Poly Vinylidene Di-Fluoride (PVDF) membranes. After blocking with 3% bovine serum albumin or 3% skim milk for 1 h, the membranes were incubated with a primary antibody overnight at 4 °C. After being washed with phosphate-buffered saline (PBS) containing 0.1% Tween 20, the membranes were reacted with an antimouse or rabbit IgG horseradish peroxidase-linked secondary antibody (1:10,000) for 1 h at room temperature. Membranes were washed with PBS three times and subjected to immunoblotting using SuperSignal West Pico Substrate (Thermo Scientific, Waltham, MA, USA) or ImmunoStar LD (Wako). Band intensities were quantitatively analyzed using ImageJ. The following antibodies were used: actin (sc-47778, Santa Cruz Biotechnology, Dallas, TX, USA), AMPK (#5832, Cell Signaling Technology, Danvers, MA, USA), p-AMPK (#2535, Cell Signaling Technology), ACC (#3662, Cell Signaling Technology), p-ACC (#11818, Cell Signaling Technology), p53 (sc-393031, Santa Cruz Biotechnology), p-p53 (#9284, Cell Signaling Technology), p70S6K1 (#2708, Cell Signaling Technology), p-p70S6K1 (#9234, Cell Signaling Technology), cleaved caspase-3 (#9664, Cell Signaling Technology), caspase-3 (#9665, Cell Signaling Technology).

Whole-cell lysates were prepared in RIPA buffer. One hundred micrograms of the protein samples was separated by SDS-PAGE (7.5%), transferred to nitrocellulose membranes, and subjected to western blotting as previously described. Blots were probed with a primary antibodies specific to iNOS (Abcam, Cambridge, UK), ACC, p-ACC, AMPK, and p-AMPK (Cell Signaling Technologies, Danvers, MA, USA). Secondary antibodies were obtained from Jackson ImmunoResearch (West Grove, PA, USA). Results were normalized to the intensity of the β-actin gene (Abcam).

RIPA lysate containing phosphatase inhibitor (Beyotime Biotechnology Co., China) was added to a centrifuge tube containing a certain amount of liver tissue and homogenized. Tissue homogenates were centrifuged at 10,000 × g for 5 min at 4°C, and the supernatants were collected. The BCA protein quantitation assay (Beyotime Biotechnology Co., China) was used to measure the total protein content of the homogenate supernatant. Equal amounts of protein from each group were loaded onto 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to PVDF membranes (Millipore, Germany). Membranes were incubated with primary antibody overnight at 4°C, and the antibodies used in this study were adiponectin (1 : 1000, Cell Signaling Technology, USA), phospho-AMP-activated protein kinase (p-AMPK) (1 : 1000, Cell Signaling Technology, USA), AMPK (1 : 500, Cell Signaling Technology, USA), SREBP-1c (1 : 1000, Santa Cruz Biotechnology, USA), p-ACC (1 : 1000, Cell Signaling Technology, USA), ACC (1 : 1000, Cell Signaling Technology, USA), and FAS (1 : 1000, Cell Signaling Technology, USA). After being washed, membranes were subsequently incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Cell Signaling Technology, USA). Protein bands were visualized with the ChemiDoc™ Touch Imaging System (Bio-Rad Laboratories, Inc., USA).