Research Article

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 2

Stat6 is required for the IL-4-mediated inhibition of Nos2 gene expression in RAW264.7 cells. (a) RAW264.7 cells were transfected with control siRNA (100 nM) or Stat6 siRNA (100 nM) for 36 hours; total RNA was then prepared for quantitative real-time RT-PCR. Each column and bar represents the mean ± SEM of three independent experiments. (b) RAW264.7 cells were transfected with siRNA, as described above, and then used to prepare total cellular lysates for a western blot analysis using an anti-Stat6 antibody. (c, d) RAW264.7 cells were transfected with control siRNA (100 nM) or Stat6 siRNA (100 nM). Thirty-six hours after transfection, the cells were either left untreated (UT) or treated with IL-4 (10 ng/mL) for 30 min prior to stimulation with IFNγ (10 ng/mL) and/or LPS (100 ng/mL) for 8 hours before the preparation of total RNA for quantitative real-time RT-PCR. The relative Nos2 mRNA expression levels are shown as percentages of the activity of cells transfected with the control siRNA and stimulated with IFNγ and LPS. Each column and bar represents the mean ± SEM of three independent experiments. The asterisks denote a statistically significant difference compared to the cultures treated with IL-4 ( , Student’s test).
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