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Figure 1. IFN-ß-induced STAT-1
activation and GAS elements in CD40 promoter are critical for IFN-ß-induced CD40 gene expression. A) RAW264.7 cells were treated with medium or IFN-ß (100 U/ml) for up to 12 h, and then total RNA was isolated and analyzed by RNase protection assay (RPA) for CD40 and GAPDH mRNA. B) RAW264.7 cells were treated in the absence or presence of IFN-ß for up to 72 h and then stained with either PE-conjugated anti-CD40 or PE-conjugated isotype-matched control antibody (Ab). Cells were subjected to FACS analysis. C) RAW264.7 cells were incubated in the absence or presence of IFN-ß (100 U/ml) for up to 4 h. Protein lysates were prepared and subjected to immunoblotting with antiphospho-STAT-1Tyr701, antiphospho-STAT-1Ser727, antiphospho-STAT-2Tyr690, stripped, and reprobed with anti-STAT-1, STAT-2, and antiactin as loading controls. D) WT and GAS mutant constructs of the human CD40 promoter. E) RAW264.7 cells were transiently transfected with 0.2 µg of indicated constructs, treated with medium or IFN-ß (100 U/ml) for 12 h, and then analyzed for luciferase activity. F) WT or STAT-1-/- primary murine microglia were treated with IFN-ß for 4 h, and then total RNA was isolated and analyzed by RPA for CD40 and GAPDH mRNA expression. Representative of 3 experiments.
