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. 2001 Jun 18;193(12):1351–1360. doi: 10.1084/jem.193.12.1351

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© 2001 The Rockefeller University Press

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/).

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A subset of NF-κB–dependent promoters is not constitutively accessible to NF-κB, but accessibility can be increased by adequate stimulation. (a) Raw 264.7 macrophages were transfected with a p65 expression vector or empty vector. Under these experimental conditions, transfected p65 is constitutively nuclear (and can activate transcription from naked cotransfected DNA). ChIP assay with an anti-p65 antibody shows that p65 is recruited only to MnSOD and MIP-2 promoters but not on the MCP-1 and RANTES promoters. Stimulation with LPS for 2 h is followed by NF-κB recruitment to the latter promoters. (b) IFN-γ pretreatment induces hyperacetylation of the MCP-1 promoter and makes it immediately accessible to NF-κB. Raw 264.7 cells were prestimulated with IFN-γ for 2 h and then stimulated with LPS for 20 or 40 min. ChIP assay was performed with anti-p65 antibody or anti-AcH4 serum. Control dilutions of IP supernatant in the anti-AcH4 ChIP are shown. Similar results were obtained with the RANTES promoter.

A subset of NF-κB–dependent promoters is not constitutively accessible to NF-κB, but accessibility can be increased by adequate stimulation. (a) Raw 264.7 macrophages were transfected with a p65 expression vector or empty vector. Under these experimental conditions, transfected p65 is constitutively nuclear (and can activate transcription from naked cotransfected DNA). ChIP assay with an anti-p65 antibody shows that p65 is recruited only to MnSOD and MIP-2 promoters but not on the MCP-1 and RANTES promoters. Stimulation with LPS for 2 h is followed by NF-κB recruitment to the latter promoters. (b) IFN-γ pretreatment induces hyperacetylation of the MCP-1 promoter and makes it immediately accessible to NF-κB. Raw 264.7 cells were prestimulated with IFN-γ for 2 h and then stimulated with LPS for 20 or 40 min. ChIP assay was performed with anti-p65 antibody or anti-AcH4 serum. Control dilutions of IP supernatant in the anti-AcH4 ChIP are shown. Similar results were obtained with the RANTES promoter.