Fas ab22759

Protein expression levels of genes involved in the lipid synthesis and antioxidant enzymes were determined by western blot analysis as previously described [48 (link)]. Nuclear fractions were used for the determination of SREBP-1 and -2, and cytosol fraction was used for the rest experiments. Briefly, sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed, and the bands were transferred to a nitrocellulose membrane. Protein expression was visualized using the enhanced chemiluminescence, equipped with CAS-400 (Core Bio, Seoul, Korea) and evaluated using ImageJ software (National Institutes of Health, Bethesda, MD, USA). The protein expression level was expressed as the ratio to α-tubulin, β-actin, or lamin B1. Primary antibody α-tubulin (ab52866), β-actin (ab8226), and FAS (ab22759) were purchased from Abcam Inc. (Cambridge, MA, USA). SOD (FL-154, sc-11407), CAT (F-17, sc-34285), GPx (B-6, sc-133160), SREBP-1 (H-160, sc-8984), SREBP-2 (H-164, sc-5603), HMGCR (H-300, sc-33827), CPT1 (sc-139482), and lamin B1 (ZL-5; sc-56145) were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA).

Quantitation of protein was carried out by Western blot assay as previously described [53 (link)]. In brief, protein was separated by sodium dodecylsulfate polyacrylamide gel and transferred to a nitrocellulose membrane (Amersham Biosciences, Uppsla, Sweden). The targeted protein band was detected using CAS-400 (Core Bio, Seoul, Korea). The calculation was performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Protein expression was normalized to that of β-actin. The primary antibodies used in this study were β-actin (ab8227) and FAS (ab22759), which were purchased from Abcam Inc. (Cambridge, UK). Phospho-AMPKα (p-AMPK, #2535) was obtained from Cell Signaling Technology (Beverly, MA, USA). SREBP-1 (sc-8984), ACC (sc-26817), PPAR-α (sc-9000), CPT1 (sc-139482), SREBP-2 (sc-5603), HMGCR (sc-33827), and CYP7A1 (sc-25536) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The secondary horseradish peroxidase-conjugated antibodies (from Abcam Inc.) were donkey anti-rabbit IgG H&L (ab6802), rabbit anti-goat IgG H&L (ab6741), and rabbit anti-mouse IgG H&L (ab6728).