Figure 3From: Division of labor between IRF1 and IRF2 in regulating different stages of transcriptional activation in cellular antiviral activitiesThe binding of IRF1 and IRF2 to the TLR3 promoter is regulated by their protein levels. A IRF1 is a strong activator whereas IRF2 is a weak activator. A luciferase reporter construct containing five GAL4 DNA binding sites was co-transfected to HeLa cells with a construct expressing either the GAL4 DNA binding domain-IRF1 fusion or the GAL4 DNA binding domain-IRF2 fusion protein for 48 hours, followed by treatment with IFN-α for 12 hours. Luciferase activity was measured as in Figure 1A. B IRF2 is expressed constitutively and IRF1 is rapidly induced by IFN-α treatment. HeLa cells were treated with IFN-α for various times as indicated above the panel. The cells were harvested and analyzed for the protein levels of IRF1, IRF2, and BRG1 using Western blotting. β actin was used as a loading control. C IRF1 and IRF2 binding to the TLR3 promoter. HeLa cells were treated with IFN-α for various times as indicated within the panel. ChIP assays were performed and analyzed using q-PCR as in Figure 2A & B. D Over-expression of IRF1 activated TLR3 promoter. pGL3-TLR3pr-luc was co-transfected with a control vector or IRF1 expression vector, pcDNA4-IRF1 into HeLa cells for 48 hours as indicated below the panel. The luciferase activity was determined as in Figure 1A. E IRF2 is required for induction of IFN-β by polyI/polyC. HeLa cells transfected with the control or siIRF2 constructs were selected in puromycin for two days. Following treatment with 20 μg/ml of polyI/polyC for 6 hours, the total RNAs were isolated and analyzed for the presence of IFN-β mRNA by real-time PCR.Back to article page