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 7
Overexpression of the coactivator CBP partially attenuates the IL-4-mediated inhibition of Nos2 promoter activity in RAW264.7 cells. (a) Analysis of endogenous BOB.1 expression in RAW264.7 cells. The cells were treated with medium alone (UT) or IFNγ (10 ng/mL) and/or LPS (100 ng/mL) for 8 hours before the preparation of nuclear extracts. Twenty micrograms of nuclear extract was analyzed by western blotting using an antibody against BOB.1 or an antibody against TATA-binding protein (TBP), which was used as a loading control. Nuclear extracts from the mouse leukemia cell line BCL1-B20 (BCL) were used as a positive control for BOB.1 expression (lane 1). (b) RAW264.7 cells were transiently co-transfected with either the empty vector or wild-type CBP expression plasmid and the pNOS-62 luciferase reporter construct. Twenty-four hours after transfection, the cells were treated with medium alone (untreated: UT) or 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 measurement of luciferase activity. The relative luciferase activities are shown as the percentage of the activity of cells transfected with the empty vector 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; ns, not significant).
