Figure 2From: Division of labor between IRF1 and IRF2 in regulating different stages of transcriptional activation in cellular antiviral activitiesIRF1 and IRF2 differentially regulate the basal and induced expression of the TLR3 gene. A IRF1 and IRF2 binding to the TLR3 promoter in the basal and stimulated states. Chromatin was prepared from HeLa cells with or without IFN-α treatment for 12 hours as in Figure 1B and subjected to immunoprecipitation using anti-IRF1 and anti-IRF2 antibodies. The ChIP DNA was analyzed using specific primers for the TLR3 promoter or for the control sequence of the β globin gene. B The ChIP DNA in panel A was quantified using real-time PCR analysis. C IRF1 and IRF2 were knocked down using small interference RNA constructs. HeLa cells were transfected with pREP4-siIRF1 or pREP4-siIRF2. The cells were harvested after selection with puromycin (1 ug/ml) for 2 days and analyzed by Western blotting. The control is a small interference RNA construct that failed to knock down IRF1. β actin was used as a protein loading control. D Knocking-down IRF1 and IRF2 inhibits the expression of the TLR3 gene. HeLa cells were transfected with a control, siIRF1, or siIRF2 and selected with puromycin for two days. Following stimulation with IFN-α for 12 hours, total RNAs were isolated and the TLR3 mRNA levels were determined using q-PCR. E Knocking-down IRF1 and IRF2 inhibits the TLR3 promoter activity. pREP4-TLR3pr-luc was co-transfected into HeLa cells with a control, or siIRF1, or siIRF2 construct for 48 hours. Following stimulation with IFN-α for 12 hours, the cells were harvested and luciferase activity determined as in Figure 1A.Back to article page