Anti-Inflammatory Cytokine Interleukin-4 Inhibits Inducible Nitric Oxide Synthase Gene Expression in the Mouse Macrophage Cell Line RAW264.7 through the Repression of Octamer-Dependent Transcription
Figure 6
Analysis of OCT DNA-binding activity in nuclear extracts from RAW264.7 cells. (a) Effect of IL-4 treatment on OCT DNA-binding activity. RAW264.7 cells were treated with medium alone or IL-4 (10 ng/mL) for 30 min prior to stimulation with IFNγ (10 ng/mL) and/or LPS (100 ng/mL) for 4 hours before the preparation of nuclear extracts. The OCT binding activity was assessed by EMSA. (b) OCT DNA-binding activity in nuclear extracts from RAW264.7 cells treated with IFNγ and LPS. RAW264.7 cells were cultured in the presence of IFNγ (10 ng/mL) and LPS (100 ng/mL) for the indicated time prior to the preparation of nuclear extracts. In total, 10 μg of each nuclear extract was analyzed for OCT binding activity by EMSA. (c) Analysis of OCT DNA-binding affinity to Nos2 OCT by an oligonucleotide competition assay. Nuclear extracts were prepared from RAW264.7 cells stimulated with IFNγ (10 ng/mL) and LPS (100 ng/mL) for 30 min. The OCT DNA-binding activity was determined by EMSA using radio-labeled OCT oligonucleotides corresponding to the Nos2 OCT (NOS OCT) site or the immunoglobulin κ chain OCT site (Igk OCT) in the presence or absence of a 25-fold excess of unlabeled wild-type (wt) or mutant (mut) oligonucleotide, as indicated. (d) Antibody super-shift assay for Nos2 OCT. Nuclear extracts (NE) from RAW264.7 cells stimulated with IFNγ (10 ng/mL) and LPS (100 ng/mL) for 30 min were incubated with the indicated antibodies (1 μg each) before analysis of the binding activity, as described above.
