Louis, MO, USA). An alkaline phosphatase-conjugated substrate Western blotting detection system kit was purchased from Bio-Rad (Hercules, CA, USA). Alkaline phosphatase-conjugated anti-mouse, anti-rabbit, and anti-goat IgG antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were diluted 1:5000 prior to use. The production of intracellular ROS
was measured using H2DCFDA as previously Everolimus nmr described . H2DCFDA reacts with ROS to form the highly fluorescent compound dichlorofluorescein. To measure ROS GF-1 cells starved by growth in low-serum Leibovitz’s L-15 medium with 1% FBS were treated with nodavirus (104 TCID50 mL−1) for 24 h followed by 10 μM H2DCFDA for 20 min. Control cells received DMSO. Cells were collected following exposure to nodavirus or DMSO, and fluorescence was determined with excitation and emission wavelengths of 485 and 520 nm, respectively, using a microplate reader (Thermo Labsystems, Waltham, MA, USA). To determine the production of ROS during nodavirus infection, cells were first incubated with the ROS scavenger, N-acetylcysteine (NAC), for 2 h, followed by nodavirus buy BMS-754807 infection. The ROS level was determined by dividing the absorbance of the infected group by the absorbance of the control group. Dityrosine can be formed by a horseradish peroxidase-catalyzed oxidation of tyrosine in the presence of hydrogen peroxide (H2O2). Ten milligrams of horseradish peroxidase was dispensed in 500 mL of 5 mM
tyrosine prepared in 0.1 M borate buffer, pH 9.1. Then, 142 μL of 30% H2O2 was added and mixed by brief swirling. After incubation at room temperature for 60 min, 175 μL of 2-mercaptoethanol was added to the reaction mixture. The resulting solution was immediately buy Atezolizumab frozen in liquid nitrogen and lyophilized. The lyophilized material was dissolved in 250 mL of distilled water and the pH was adjusted to 8.8 with a
few drops of 0.01 M NaOH. The resulting solution was loaded onto a DEAE column that has been pre-equilibrated with 20 μM NaHCO3, pH 8.8, and was eluted using 200 μM borate buffer, pH 8.8. The dityrosine-containing solutions were pooled and lyophilized. The dityrosine produced in the mixture was chromatographically purified. To do this, the supernatant was loaded onto a BioGel P-2 column equilibrated with 100 mM NH4HCO3. The column was eluted with 100 mM NH4HCO3 with a flow rate 40 mL/h. The lyophilized dityrosine was dissolved in 20 mL of 100 mM formic acid and the pH was adjusted to 2.5 by adding concentrated formic acid. The column was eluted with 100 mM formic acid, and the dityrosine-containing solution was lyophilized and stored at −20 °C . An isocratic reverse-phase HPLC system also was used to analyze dityrosine in conjunction with both absorbance detection systems. The 4.6×250 mm2 reverse-phase column (ODS II Spherisorb; LC-Resources, Deerfield, IL, USA) has an 11% carbon loading and a particle size of 5 mm. The solvent consisted of 92% water, 8% acetonitrile, and 0.1% trifluoroacetic acid.