A0945

Liver samples were lysed using lysis buffer (50 mmol/L Tris, 150 mmol/L NaCl, 1 mmol/L EDTA, 1% Triton X-100) supplemented with protease inhibitors (1 μg/mL leupeptin, 1 μg/mL aprotinin, 1 μg/mL pepstatin, and 50 μg/mL polymethyl sulfonyl fluoride). Equal amounts of tissue lysate were resolved using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins were then transferred onto polyvinylidene fluoride membranes (16916600, Roche), blocked with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween 20 for 1 h at room temperature, and probed with primary antibodies against acyl-coenzyme A oxidase 1 (ACOX1) (AB091), long-chain fatty acid CoA ligase 1 (ACSL1) (A1000), short-chain acyl-CoA dehydrogenase (ACADS) (A0945), carnitine palmitoyl transferase 1 (CPT1) (A5307), and β-actin (A026) (all from ABclonal Technology). The specific proteins were detected using horseradish peroxidase-conjugated secondary antibodies (sc-2004, Santa Cruz Biotechnology), developed with SuperSignal West Pico Chemiluminescent Substrate (34,080, Thermo Scientific), and visualized using a Kodak Image Station 2000 MM. Immunoblotting results were quantified using Image J software (1.49 s). All the immunoblotting assays were performed using three biologic replicates.

HepG2 cells were lysed using lysis buffer (50 mM Tris, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100) supplemented with protease inhibitors (1 μg/mL Leupeptin, 1 μg/mL Aprotinin, 1 μg/mL Pepstatin, and 50 μg/mL PMSF). Equal amounts of cell lysates were resolved by 10% SDS-PAGE. Proteins were then transferred onto PVDF membranes (16916600, Roche), blocked with 5% non-fat milk in Tris-buffered saline containing 0.05% Tween 20 for 1 hour, and probed with primary antibodies against ADH1C (A8081, ABclonal Technology), ALDH1A1 (A1802, ABclonal Technology), ACSL1 (A1000, ABclonal Technology), ACOX1 (A8091, ABclonal Technology), ACADS (A0945, ABclonal Technology), and β-Actin (4967L, Cell Signaling Technology). The specific proteins were detected with HRP-conjugated secondary antibodies (sc-2004, Santa Cruz Biotechnology), developed with SuperSignal West Pico Chemiluminescent Substrate (34080, Thermo Scientific) and visualized by Kodak Image Station 2000 MM. Immunoblotting results were quantified using Image J software (1.49s). All the immunoblotting assays were performed in three biological replicates.