Peroxynitrite

Nitration was carried out as previously described using Peroxynitrite (21 (link), 22 (link)). Medin was exchanged into nitration buffer (100 mM potassium phosphate, 25 mM sodium bicarbonate, pH 7.4, 0.1 mM diethylenetriaminepentaacetic acid). Peroxynitrite (Cayman Chemical, Ann Arbor, MI) was added to the required final molar ratio (0.5–50 excess for dot-blot, 10 excess for all other experiments) and vortexed. Nitration was monitored by measuring an increase in absorbance at 430 nm.

Peroxynitrite was purchased from Cayman, USA, and the stock solution concentration was determined by absorbance at 302 nm in NaOH (ε_302 nm=1670 M⁻¹ cm⁻¹) [3] (link). Control cells were treated with decomposed Peroxynitrite.

Immortalized rat intestinal epithelial cells (IEC-6) were purchased from ATCC (CRL-1592) was maintained in high glucose Dulbelcos modified eagles medium (DMEM), Corning (Tewksbury, MA) supplemented with 10% fetal bovine serum (FBS), Atlanta biologicals (Norcross, GA) suplimented with 2 mM glutamine, 0.1 U/ml Insulin, 100 U/ml Penicillin, and 100 μg/ml streptomycin; Gibco (Grand Island, NY) at 37 °C in a humidified atmosphere of 5% CO₂. The cells were then treated with recombinant rat HMGB1 (Mybiosource. San Diego, CA) 30 ng/ml a cytokine mediator of inflammation released by activated macrophages able to bind to and activate RAGE & TLR4, Apocynin (Sigma-Aldrich) 100 µM an inhibitor of NADPH oxidase activity and thus preventing production of superoxide, RAGE antagonist (FPS-ZM1, Milipore, MA) 200 nM V-domain ligand binding RAGE inhibitor, Peroxynitrite (Cayman Chem, Ann Arbor, MI) 300 µM a highly reactive structural isomer of nitrate, Phenyl Boronic Acid (FBA) (Sigma Aldrich) 100 µM was used as a scavenger for Peroxynitrite, PI3K inhibitor (LY294002, Sigma Aldrich) 10 µM as an inhibitor for AKT phosphorylation as required.