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. 2000 Aug 21;192(4):581–586. doi: 10.1084/jem.192.4.581

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© 2000 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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graphic file with name JEM000151.f1a.jpg — Murine TNF-α gene expression is dependent upon NFAT. (A) −200 nucleotides upstream of the TNF-α mRNA cap site are sufficient for CsA-sensitive anti-CD3 induction of TNF-α in T cells. The Ar-5 T cell clone was transfected with CAT reporters linked to murine TNF-α promoters containing −200, −475, or −982 nucleotides upstream of the mRNA cap site and treated with anti-CD3 antibody and pretreated with CsA for 10 min as indicated. A representative CAT assay of three independent transfections is shown. Transfections included CMV–β-gal as a control and CAT activity was normalized to β-galactosidase activity. (B) NFATp binds to six sites in the TNF-α promoter. Quantitative DNaseI footprinting using the murine TNF-α promoter (−200 to +87 nucleotides relative to the transcription start site) and increasing concentrations of recombinant NFATp (100 ng, 400 ng, and 2 μg) or BSA at 2 μg (–). The NFATp binding sites and the ATF-2/Jun binding site (CRE) are indicated in the drawing at the right.

graphic file with name JEM000151.f1b.jpg — Murine TNF-α gene expression is dependent upon NFAT. (A) −200 nucleotides upstream of the TNF-α mRNA cap site are sufficient for CsA-sensitive anti-CD3 induction of TNF-α in T cells. The Ar-5 T cell clone was transfected with CAT reporters linked to murine TNF-α promoters containing −200, −475, or −982 nucleotides upstream of the mRNA cap site and treated with anti-CD3 antibody and pretreated with CsA for 10 min as indicated. A representative CAT assay of three independent transfections is shown. Transfections included CMV–β-gal as a control and CAT activity was normalized to β-galactosidase activity. (B) NFATp binds to six sites in the TNF-α promoter. Quantitative DNaseI footprinting using the murine TNF-α promoter (−200 to +87 nucleotides relative to the transcription start site) and increasing concentrations of recombinant NFATp (100 ng, 400 ng, and 2 μg) or BSA at 2 μg (–). The NFATp binding sites and the ATF-2/Jun binding site (CRE) are indicated in the drawing at the right.