Inducible recruitment of IRF-3 to the endogenous IL-12p35 locus in human DCs (ChIP assay). Monocyte-derived DCs were either incubated with medium alone (lanes 2, 6, and 10) or stimulated with LPS (1 μg/mL; lanes 3, 7, and 11), PolyI:C (10 μg/mL; lanes 4, 8, and 12), or Pam3CSK4 (10 μg/mL; lanes 5, 9, and 13). After 3 hours, cells were treated with 1% formaldehyde to cross-link proteins bound to DNA. After sonication, chromatin samples were immunoprecipitated with anti-IRF-3 rabbit polyclonal antibodies (lanes 6-9) or control antibodies (lanes 2-5). Purified DNA was amplified using primers specific for IL-12p35, IL-12p40, or GAPDH promoter regions. For each sample, 2% of the cross-link-released chromatin was saved and used as input control (lanes 10-13). Lane 1 indicates PCR reaction without sample.
