Chromatin Immunoprecipitation (ChIP) Assay Kit

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	ChIP and EMSA were performed as previously described

Protocol tips
ChIP and EMSA were performed as previously described

Publication protocol

ChIP and EMSA were performed as previously described48. The association of exogenous NF-κB with ANT1 promoter in HEK293 cells was confirmed using a chromatin immunoprecipitation assay kit # 17-295, Millipore) following the manufacturer’s protocol. Transfected by NF-κB/p65 plasmid, cells each about 5 × 106 cells) were cross-linked by formaldehyde (final concentration of 1%) for 10 min at 37 °C, and then washed by cold PBS twice. The cells were centrifuged and pellets were lysed by 100 μl 1% SDS lysis buffer and sheared by sonication. The sonicated cell supernatant was then diluted by 9 fold (900 μl) ChIP dilution buffer and 1/50 (20 μl) was accepted as input. After the cross-linked proteins and DNA pulled down with p65 antibody (1:100, #8242, CST) or normal IgG as a negative control overnight at 4 degree, the cross links (both input and immunoprecipitated group) were reversed and DNA was recovered by phenol/chloroform extraction. The extract ed DNA was re-dissolved in 20 μl PCR-grade water. 1 μl of input or immunoprecipitated DNA were used as templates and confirmed by PCR (35 cycles) using following promoter primers (ChIP-F:5′-CACCTGCCCAGCCAATGC-3′ and ChIP-R: 5′-CGCAGGCAGCCCGTTCGT-3′). Products of ChIP-PCR were separated on a 1% agarose gel with ethidium bromide. Immunoprecipitation of proteins, after ChIP with antibodies against NF-κB/p65, was confirmed by Western blot analysis before the ChIP-PCR analysis. For EMSA assay, infrared dye-labeled probe (50 nM) were used in respective incubation and the three sense sequences of ANT1 NREs were 5′-AAGGGGGAGCTGCGGGCCAG (NRE1), 5′-GCGGCGGCCCCCTAGCGTCG (NRE2) and 5′-CGCAGGGTCGGGGACTGCGCG (NRE3). Consensus NRE and mutant NRE were 5′-AGTTGAGGGGACTTTCCCAGGC, 5′-CAAAAATGTTCAAAAATGTT.

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