IRF8 positively regulates IFN-β transcription in monocytes. (A) Primary monocytes were transfected with IRF8 siRNA or control siRNA. Western blot (bottom) shows the IRF8 protein level in total cell lysates. IFN-β transcription in monocytes stimulated with LPS for 2 hours, determined by real-time PCR (top). (B) The mapping of IRF8-binding sites across IFN-β genomic DNA using 5 different pairs of primers (bottom). Primary monocytes were stimulated with LPS for 1 hour before ChIP analysis. ChIP was performed using either anti-IRF8 or control anti-GST antibody. (A-B) Similar results were obtained in 4 independent experiments with monocytes from 4 different donors, one of which is shown. (C) The sequences of IFN-β WT_{(−99 to −71)} probe containing the EIRE motif and WT_{(−83 to −55)} probe containing the EICE motif are shown. (D) EMSA was performed with IFN-β WT_{(−99 to −71)} probe (lanes 1-4) or WT_{(−83 to −55)} probe (lanes 5-8). Lanes 1 and 5 indicate free probe; lanes 2 and 6, with nuclear extract from LPS stimulated THP-1 cells after 2 hours; lanes 3 and 7, addition of 20-fold molar excess of unlabeled WT_{(−99 to −71)} competitor probe; and lanes 4 and 8, addition of 20-fold molar excess of unlabeled WT_{(−83 to −55)} competitor probe. (E) EMSA was performed with IFN-β WT_{(−83 to −55)} probe. Lane 1 indicates free probe; lanes 2 to 4, nuclear extract from LPS stimulated THP-1 cells after 2 hours; lane 3, control anti-GST antibody; and lane 4, anti-IRF8 antibody. (D-E) Similar results were obtained in 4 independent experiments, one of which is shown.