ChIP Assay for Transcription Factor Binding

ChIP assay was performed according to the protocol specified by the manufacturer (HighCell# ChIP kit, Diagenode Inc.), In brief, treated cells were collected by trypsinisation, and DNA-protein interactions were cross-linked with 1% formaldehyde. Chromatin shearing was performed by sonication at 4°C with a Bioruptor® Plus equipped with a Minichiller (Diagenode Inc.). Lysates were centrifuged, and aliquots (1%) of the supernatants were collected for input control, while the remaining supernatants were incubated overnight (at 4°C) with magnetic beads coated with ChIP grade antibodies against p65, AhR or Arnt. DNA was isolated from the collected precipitates after reversal of the cross-linking by incubation for 15 min at 55°C. Real-time PCR were performed using primer sets for human CXCL8 and CCL5 promoter regions containing NF-κB response elements [62 (link)],[63 (link)]. The sequences of primers used for ChIP assay were as follows: human CXCL8 sense 5’-AGTGTGATGACTCAGGTTTGCCCT-3’ and anti-sense 5’-AAGCTTGTGTGCTCTGCTGTCTCT-3’; human CCL5 promoter sense 5’-GGGAAGAAGATTGCCTAAAC-3’ and antisense 5’-TGTGGAAATCAAAGGGACAG-3’.