Occupancy of the IFN-β promoter by IRF8 is stabilized by PU.1. (A) ChIP analysis (with PU.1 antibody or control GST antibody) was performed in unstimulated and LPS-stimulated primary monocytes. ZNF524 primer was used as control. (B) Primary monocytes were transfected with PU.1 siRNA or control siRNA. Western blot (bottom) shows the PU.1 protein level in total cell lysates. Top: IFN-β gene expression in cells that were stimulated with LPS for 2 hours. (A-B) Similar results were obtained in 5 independent experiments with monocytes from 5 different donors, one of which is shown. (C) 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 GST antibody; and lane 4, PU.1 antibody. (D) The sequences of IFN-β wild-type WT_{(−83 to −55)} probe and mutant M-83_{(−83 to −55)} and mutant M-70_{(−83 to −55)} probes are shown. (E) EMSA was performed with nuclear extracts from HEK293 cells transfected with mock control (lanes 1-3), IRF8 plasmid (lanes 4-6), PU.1 plasmid (lanes 7-9), or both IRF8 and PU.1 plasmids (lanes 10-12). WT_{(−83 to −55)} probe and M-83_{(−83 to −55)} and M-70_{(−83 to −55)} probes were used. (C,E) Similar results were obtained in 3 independent experiments, one of which is shown.