Anti adiponectin

The proteins in the cells were extracted with Pro-Prep (Intron Biotechnology, Seongnam, Korea) and centrifuged after sonication. A total of 20 μg of each protein was separated with SDS-polyacrylamide gel electrophoresis (PAGE). After the size-dependent separation, the proteins were transferred to polyvinylidene difluoride (PVDF) membranes using semi-dry transfer (Bio-Rad, Hercules, CA, USA). The membranes were incubated with primary antibodies overnight at 4 °C, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (Abcam, Cambridge, UK) for 1 h at room temperature. The signals were detected with chemiluminescence reagents (Abclon). Anti-acetyl histone H3 (Merck Millipore, 06-599, Burlington, MA, USA), anti-histone H3 (Santa Cruz Biotechnology, SC-10809, Dallas, TX, USA), anti-acetylated α tubulin (Santa Cruz Biotechnology, SC-23950), anti-tubulin (Santa Cruz Biotechnology, SC-32293), anti-Runx2 (Abcam, ab23981), and anti-Adiponectin (Cell Signaling Technology, #2789, Danvers, MA, USA) were used for the immunoblotting assay in this study. The levels of acetyl H3 and acetylated α tubulin were quantified with ImageJ and normalized to the quantified levels of H3 and α tubulin.

The proteins were extracted from the retinal homogenates or cells using radioimmunoprecipitation assay (RIPA) lysis buffer, which contained 0.5 M Tris-HCl (pH 7.4), 1.5 M NaCl, 2.5% deoxycholic acid, 10% NP-40, 10 mM EDTA, and 10% protease inhibitors (Complete Mini; Roche Diagnostics Corp., Indianapolis, IN, USA). For the western blot analysis, the protein samples were separated using a 10% sodium dodecyl sulfate- (SDS-) polyacrylamide gel and transferred to a polyvinylidene difluoride membrane (Immobilon-P; Millipore Corp., Billerica, MA, USA). The analysis was performed using anti-adiponectin (Cell Signaling Technology Inc. for the rat and cell samples at a 1 : 1000 dilution), anti-adiponectin receptor 1 (Santa Cruz Biotechnology Inc., for the rat samples at a 1 : 500 dilution; Epitomics, Inc., Burlingame, CA, USA, for the cell samples at a 1 : 500 dilution), anti-adiponectin receptor 2 (Santa Cruz Biotechnology Inc., for the rat samples at a 1 : 500 dilution; Bioss Inc., Woburn, MA, USA, for the cell samples at a 1 : 2000 dilution), or anti-β-actin antibodies (Bioss Inc., Woburn, MA, USA, for all of the samples at a 1 : 5000 dilution). Immunodetection was performed by enhanced chemiluminescence (Pierce Biotechnology, Rockford, IL, USA) according to the manufacturer's instructions. Protein levels were determined using densitometry analysis of the protein bands.

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).