Anti acc

HepG2 cells were seeded in a 35-mm culture dish with a density of approximately 1 × 10⁶ cells/dish and allowed 24 h to attach. Cells were harvested and lyzed by M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific, USA) containing the proteinase inhibitor cocktail (Thermo Fisher Scientific, USA). Protein from cell lysate was collected and concentration quantified by BCA Assay Reagent (Thermo Fisher Scientific, USA). Equal amounts of proteins per lane were separated by 8–12% (SDS) polyacrylamide gel electrophoresis and transferred to PVDF membranes. Then, the membranes were incubated with RAPIDBLOCK solution (Thermo Fisher Scientific, USA). Membranes were then incubated with anti-FASN (Abcam, USA), anti-ACC (Merck Millipore, USA), and anti-ACLY (Cell Signaling Technology, USA) and then exposed to horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Life Technologies, Invitrogen). β-actin was used as an internal control (Cell Signaling Technology, USA). Finally, protein bands were visualized by LuminataTM Forte Western HRP Substrate (Merck Millipore, USA) and detected by CCD camera (Chemiluminescence Image Quant LAS 4000; GE Healthcare Life Sciences, Pittsburgh, PA, USA). Percentages of relative expression levels of protein/actin were calculated by Image J software version 1.46.

Capsaicin and C75 were obtained from Sigma Chemical Co. (St. Louise, MO, USA). Eagle's Minimum Essential Medium (EMEM), phosphate-buffered saline (PBS), fetal bovine serum (FBS), penicillin-streptomycin, trypsin-EDTA, sodium pyruvate, L-glutamine, sodium bicarbonate solution, non-essential amino acid solution, and PBS tablets were obtained from Gibco BRL (Grand Island, NY, USA). Propidium iodide molecular probe were purchased from Life Technology (Invitrogen, Grand Island, NY, USA). M-PER mammalian protein extraction reagent, BCA protein assay reagent (bicinchoninic acid), polyvinylidenedifluoride membranes (PVDF) membrane, and Halt Protease Inhibitor Cocktail were purchased from Thermo Scientific (Rockford, IL, USA). Anti-fatty acid synthase (FASN) and anti-β-actin antibodies were purchased from Abcam (Biomed Diagnostics (TH) Co., Ltd, Thailand). Anti-ACC and anti-ACLY were purchased from Merck Millipore (Darmstadt, Germany) and Cell Signaling Technology Inc (Boston, MA, USA), respectively. All other chemicals and reagents were the highest grade available.