Figure 5: NO induces nitration of tyrosine residues of IRF5 protein in macrophages.

(a) RAW264.7 macrophages were pretreated with SNAP (500 μM) for 30 min and the cells were then activated with IFN-γ (10 ng ml⁻¹) for various time intervals (10, 20, 30 and 60 min). Western blotting was performed for the analysis of STAT1 phosphorylation. β-Actin was used as a control. (b) RAW264.7 macrophages were pretreated with SNAP (500 μM) for 30 min, and the cells were then activated with IFN-γ (10 ng ml⁻¹) for various time intervals (10, 20, 30 and 60 min). The cytosolic fraction and nuclear fraction of protein was prepared, and western blotting was performed for the analysis of phosphorylated STAT1 and STAT1 protein. (c) BMDMs from WT mice were stimulated with IFN-γ (10 ng ml⁻¹) and LPS (200 ng ml⁻¹) in the presence of SNAP (500 μM) for 6 h, followed by ChIP assay. Three micrograms of an anti- phosphorylated STAT1 antibody or isotype-matched IgG as control antibody was used in the immunoprecipitation step. PCR was used to quantify the amount of precipitated DNA with primers flanking the STAT1-binding site of the iNOS promoter region. Each bar represents mean±s.d. from three independent experiments, unpaired Student’s t-test, *P<0.05, versus normal IgG. (d) BMDMs from WT and iNOS^−/− mice were stimulated with IFN-γ (10 ng ml⁻¹) and LPS (200 ng ml⁻¹) in the presence of SNAP (500 μM) or L-NIL (40 μM) for 24 h. The cell lysates were then immunoprecipitated with an anti-nitrotyrosine antibody and blotted with an anti-IRF5 antibody. Data are representative of three independent experiments. (e) The 293T cells were transfected with IRF5 plasmid for 40 h in the presence of SNAP (500 μM) or L-NIL (40 μM). Cell lysates were immunoprecipitated with an anti-nitrotyrosine antibody and immunoblotted with an anti-IRF5 antibody. (f) The 293T cells were cotransfected with an IL-12 promoter reporter construct and an IRF5 plasmid in the presence of SNAP (100, 200 and 500 μM) for 30 h. Luciferase assays were performed and luciferase activities were normalized to β-galactosidase activity. Each bar represents mean±s.d. from three independent experiments, unpaired Student’s t-test, *P<0.05 and ***P<0.001 versus IRF5 plasmid only.