Anti nitrotyrosine

All of the drugs, including urethane, fructose, resveratrol, compound C, and dimethyl sulfoxide (DMSO), and the mouse anti-actin, goat anti-rabbit, and goat anti-mouse IgG secondary antibodies were obtained from Sigma-Aldrich (Sigma Chemical Co., St. Louis, MO, USA). Anti-p-AMPK^T172, anti-AMPK, anti-ACC^S79, anti-p-ERK^T202/Y204, anti-ERK, anti-nNOS^S1416, anti-nNOS, anti-eNOS^S177, anti-eNOS, anti-iNOS, anti-p-RSK^T359/S363, and anti-RSK antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-p22-phox and anti-nitrotyrosine were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-p47-phox and anti-p67-phox were purchased from Millipore (Bedford, MA, USA). Anti-Cu/Zn-SOD and anti-Mn-SOD were obtained from StressGen Biotechnologies (La Jolla, CA, USA) and Abcam (Cambridge, UK), respectively.

The expressions of Bax, Bcl-XL, inducible nitric oxide synthase (iNOS), and nitrotyrosine were determined by immunoblotting of the renal extracts. Tissue was lysed in 500 μL of lysis buffer containing 1 : 100 protease inhibitor cocktail and centrifuged at 14,000 rpm for 30 min. Protein concentration in the supernatant was measured (Bio-Rad, USA) and 25 μg of each sample was loaded on 8–15% SDS–PAGE gels and electrophoresed. The proteins were transferred to nitrocellulose sheets. After washing and blocking, the membranes were incubated at 4°C with the following primary antibodies: anti-nitrotyrosine (Santa Cruz Biotechnology, USA, 1 : 100), anti-iNOS (Cell Signaling Technology, USA, 1 : 1,000), and anti-Bax and anti-Bcl-XL (Cell Signaling Technology, USA, 1 : 1,000). The secondary antibody, horseradish peroxidase-conjugated goat anti-rabbit IgG (KPL, USA) or horseradish peroxidase-conjugated goat anti-mouse IgG (KPL, USA), was then added at room temperature (15–20°C) for 1 h. The blots were developed using a chemiluminescent reagent system (SuperSignal West Pico Chemiluminescent Substrate; Thermo Fisher, USA) and the images were recorded on film.

The paraffin-embedded kidney sections were then stained with a periodic acid-Schiff (PAS) reagent. Immunohistochemistry for anti-F4/80 (1 : 200; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), anti-nitrotyrosine (1 : 200; Santa Cruz Biotechnology), anti-NADPH oxidase 2 (NOX2, 1 : 500), and anti-NADPH oxidase 4 (NOX4, 1 : 300) antibodies was performed. A Zeiss microscope having an Axio cam HRC digital camera and software (Carl Zeiss, Thornwood, NY, USA) was utilized to take the images.

Serum insulin levels were measured using the ultra-sensitive rat insulin ELISA kit (Crystal Chem Inc., Downers Grove, IL, USA), and other serum-specific markers were estimated with the Lab Genomics Clinical Laboratories kit (LabGenomics, Seoul, Korea) according to the manufacturer instructions.
The pancreas tissue obtained from the experimental rats was collected, fixed in 4% paraformaldehyde, and processed for paraffin sectioning. Tissues were sectioned at a 4-μm thickness and stained with hematoxylin and eosin (H & E) to assess pathological changes of islet size under a light microscope with IMT i-solution software. Immunohistochemistry was performed with anti-insulin (1:200) and anti-nitrotyrosine (1:200) antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and observed using the laser-scanning confocal microscope (Zeiss LSM 510).

Whole lysates were obtained with RIPA buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1% Triton X-100) with freshly added protease inhibitor cocktail tablet (Roche); the protein concentrations were measured by using the Pierce® BCA Protein Assay Kit (23225, Thermo Fisher Scientific, Waltham, MA, USA). The nitrocellulose membranes (1620112, Bio-Rad) were subsequently blocked in TBST containing 5% skimmed milk powder for 90 minutes at room temperature and incubated with anti-EZH2 (Peninsula Laboratories Inc.), anti-SOD1 (Santa Cruz Biotechnology), anti–nitrotyrosine (Santa Cruz Biotechnology), and anti-β-actin (Sigma) overnight at 4°C. On following day, the membranes were incubated with secondary antibodies, respectively, and the signals were visualized with a SignalFire(tm) ECL Reagent (6883S, Cell Signaling Technology, USA).

Total proteins were extracted from cultured cells or homogenized renal tissues using radioimmunoprecipitation assay (RIPA) buffer (Sigma), and western blotting analysis was performed as described previously18 (link). The following primary antibodies were used: rabbit polyclonal anti-Sirt1 (1:800; Sigma), anti-cleaved caspase-3 (1:1000; Cell Signaling Technology, Danvers, MA), anti-nitrotyrosine (1:1000; Santa Cruz), anti-AT1R (1:500; Millipore), anti-AT2R (1:500; Millipore), anti-NF-κB (p65) (1:5000; Enzo Life Science, Farmingdale, NY, USA), anti-acetyl (K310)-NF-kB p65 (1:1000; Abcam, Cambridge, MA), anti-phospho (Ser32)-IκBα (1:1000; Cell Signaling), anti-connective tissue growth factor (CTGF; 1:1000; Peprotech, Rocky Hill, NJ, USA), hamster monoclonal anti-monocyte chemotactic protein 1 (MCP-1; 1:500; Abcam), mouse monoclonal anti-fibronectin (1:400; Abcam), anti-GAPDH (1:2000; GenScript, Piscataway, NJ, USA), and anti-proliferating cell nuclear antigen (PCNA, 1:1000; GeneTex Inc., Irvine, CA). The secondary horseradish peroxidase (HRP)-conjugated antibodies included goat anti-rabbit IgG (Santa Cruz Biotechnology), goat anti-mouse IgG (PerkinElmer, Waltham, MA, USA), and goat anti-hamster IgG (Invitrogen) diluted 1:5000. Specific signals were visualized using Immobilon Western Chemiluminescent HRP Substrate (Millipore).