Mouse anti fasn

Total protein from cells was extracted using radioimmunoprecipitation assay lysis buffer (Invitrogen; Thermo Fisher Scientific, Inc.) containing 60 µg/ml phenylmethylsulfonyl fluoride, according to the manufacturer's protocol. Protein concentration was determined using the Bradford assay. Proteins (10 µg) were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membranes were probed with the following primary antibodies overnight at 4°C: mouse anti-NF-YA (1:500; catalog no. sc-17753; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), mouse anti-FASN (1:500; catalog no. sc-55580; Santa Cruz Biotechnology, Inc.) and mouse anti-β-actin antibody (1:2,000; catalog no. sc-47778; Santa Cruz Biotechnology, Inc.). Following incubation with a goat anti-mouse horseradish peroxidase-conjugated secondary antibody (1:5,000; catalog no. 610-103-043; Rockland Immunochemicals, Inc., Pottstown, PA, USA) for 1.5 h at room temperature, immunoreactive bands were visualized using an Enhanced Chemiluminescence reagent (Thermo Fisher Scientific, Inc.). The intensity of western blot bands was measured using ImageJ software version 1.48 (National Institutes of Health, Bethesda, MD, USA). Six independent experiments were performed over multiple days.