C ebp γ

Gel shift assays were carried out in a reaction mixture containing 10 µg of nuclear extract protein, 2 µL of 10× binding buffer (200 mM Tris–HCL pH 7.6, 500 mM KCL, 10 mM MgCl₂, 10 mM DTT, 2 mM EDTA, 0.1% (v/v), Triton X-100, 50% (v/v) Glycerol, and 5 mM spermidine), 2.5 µL poly dI·dC (1µg/L), and nuclease-free water in a total volume of 19 µL. After 10-min incubation at room temperature, 1 µL of ³²P-labeled oligonucleotides (40 fmols, approximately 50,000 cpm) was then added to the reaction and incubated at room temperature for 45 min before loading to the gel. The resulting DNA–protein complex was separated from free probe by electrophoresis on a 6% polyacrylamide gel with 0.5× TBE buffer. For binding competition analysis, non-radioactive oligonucleotides (50×–100× molar excess) were added as competitors to the binding reactions as indicated in the figure legends (Table 1). For antibody interaction studies, antibodies against C/EBP-α, C/EBP-β, C/EBP-γ, C/EBP-δ, and PPAR-α (Santa Cruz Biotechnology, Santa Cruz, CA 95060) were added into the reaction mixture during the pre-incubation time. The electrophoresis was performed at 4 °C, 240 V for 2.5 h. The gel was then dried and visualized following autoradiography for 16 h at − 80 °C without an intensify screen. The densitometry quantification was performed using a Bio-Rad Image Densitometer (Model GS-700).

Carnosic acid was purchased from Cayman Chemical (Ann Arbor, MI, USA). Fetal bovine serum (FBS) was obtained from Biowest (Riverside, MO, USA). Dulbecco`s modified Eagle`s medium (DMEM), bovine calf serum (BCS), Fetal bovine serum (FBS), trypsin-EDTA, phosphate-buffered saline (PBS, pH 7.4), and Hank`s balanced salt solution (HBSS) were purchased from Welgene Inc. (Gyeongsan, Korea). Dimethylsulfoxide (DMSO), antibiotic-antimycotic solution 100X, insulin, 3-isobutyl-1-methyl-xanthine (IBMX), dexamethasone, isopropanol, formaldehyde, DCFH-DA, DHE, protease inhibitor, phenylmethanesulfonyl fluoride (PMSF), Triton X-100, 1,4-dithiothreitol (DTT), skim milk, and DPAI (4′,6-diamidino-2-phenylindole) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies of Nox4, p47^phox, p22^phox, NF-κB (p65), phospho-NF-κB (p65), IκBα, phospho-IκBα, C/EBPα, C/EBPβ, C/EBPγ, HO-1, γ-GCSc, GST, and Nrf2 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-adiponectin was obtained from Cell Signaling Technology (Beverly, MA, USA). 3T3-L1 cells were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA).