Acox1

The samples were lysed using radioimmunoprecipitation assay buffer containing protease inhibitors (Sigma-Aldrich, St. Louis, MO, USA). Protein concentrations were determined using bicinchoninic acid protein assay (G-Biosciences, Maryland Heights, MO, USA). Thirty µg of protein lysates were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transfer onto polyvinylidene fluoride membrane (Millipore, Burlington, MA, USA). The membranes were blocked at room temperature for one hour using TBS-T (10 mM Tris, 150 mM NaCl, and 0.05% Tween 20, (pH 7.6)) containing 10% skimmed milk, then probed with 1:1000 primary antibodies, such as FAS (Abcam, Cambridge, UK), ACC (Cell signaling, Danvers, MA, USA), SREBP1 (Novus, Centennial, CO, USA), ACOX1, CPT2 (Affinity Biosciences, Pottstown, PA, USA), PPARα (GeneTex, Alton Pkwy Irvine, CA, USA), HMGCR (Abcam, Cambridge, UK) and actin (Abnova, Taipei, Taiwan) at 4 °C overnight. Blots were then washed with TBS-T and incubated with a 1:5000 dilution of horseradish peroxidase-conjugated secondary antibodies (Abcam, Cambridge, UK) at room temperature for one hour. Protein bands were visualized using a chemiluminescence horseradish peroxidase substrate (Millipore, Burlington, MA, USA), and the relative signal intensity was quantified using ImageJ software.

Ficus hirta Vahl. was bought from Kangmei Pharmaceutical Co., Ltd. (Guangdong, China); palmitic acid (PA, P9697) was bought from Sigma-Aldrich (St. Louis, MO, USA); primary antibodies against IL-6 (DF6078, 1 : 1500), IL-1β (AF5103, 1 : 1500), TNF-α (AF7014, 1 : 500), SREBP-1 (AF6283, 1 : 1500), CPT1α (DF12004, 1 : 1500), ACOX1 (DF12046, 1 : 1500), and GAPDH (AF7012, 1 : 2000) were purchased from Affinity Biosciences Co., Ltd. (Jiangsu, China); antibody against CD36 (18836-1-AP, 1 : 1000) was bought from Proteintech (Wuhan, China); HMGCR (ab171830, 1 : 5000) was purchased from Abcam (Shanghai, China); RIPA buffer and BCA protein assay kit were bought from Beyotime Biotechnology (Shanghai, China); the high-fat diets (HF60), including 60% fat, 20% carbohydrate, and 20% protein, were purchased from Dyets (Dyets Biotechnology Co., Ltd., USA); the normal fat diets including 20 kcal % fat were purchased from Guangdong Medical Laboratory (Guangzhou, China); and D-(+)-glucose (CAS: 50-99-7) and fructose (CAS: 7660-25-5) were bought from Macklin Biochemical Co., Ltd. (Shanghai, China).