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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

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graphic file with name JEM000151.f3a.jpg — Kinetics of induction of cytokine mRNAs during primary in vitro stimulation of NFATp^+/+ and NFATp^−/− splenocytes. (A and B) The kinetics of induction of TNF-α by anti-CD3 are delayed in NFATp^−/− mice. Spleen cells isolated from NFATp^+/+ (+/+) and NFAT^−/− (−/−) mice were unstimulated (UN) or stimulated up to 45 h with plate-bound anti-CD3 as indicated. At the indicated times, cells were harvested and the total cellular RNA was analyzed for TNF-α, IL-4, IL-2, and L-32 mRNA levels by RNase Protection assay. (C) Lack of binding of NFATp to DNA in NFATp^−/− splenocytes. Nuclear extracts were prepared from purified splenocytes from NFATp^+/+ (+/+) and NFAT^−/− (−/−) mice that were unstimulated (UN) or stimulated with plate-bound anti-CD3 antibody for 1 h. An oligonucleotide probe matching the −76 NFAT binding site was used and the binding assay was performed in the presence or absence of the NFATp or NFATc antibodies as indicated.

graphic file with name JEM000151.f3b.jpg — Kinetics of induction of cytokine mRNAs during primary in vitro stimulation of NFATp^+/+ and NFATp^−/− splenocytes. (A and B) The kinetics of induction of TNF-α by anti-CD3 are delayed in NFATp^−/− mice. Spleen cells isolated from NFATp^+/+ (+/+) and NFAT^−/− (−/−) mice were unstimulated (UN) or stimulated up to 45 h with plate-bound anti-CD3 as indicated. At the indicated times, cells were harvested and the total cellular RNA was analyzed for TNF-α, IL-4, IL-2, and L-32 mRNA levels by RNase Protection assay. (C) Lack of binding of NFATp to DNA in NFATp^−/− splenocytes. Nuclear extracts were prepared from purified splenocytes from NFATp^+/+ (+/+) and NFAT^−/− (−/−) mice that were unstimulated (UN) or stimulated with plate-bound anti-CD3 antibody for 1 h. An oligonucleotide probe matching the −76 NFAT binding site was used and the binding assay was performed in the presence or absence of the NFATp or NFATc antibodies as indicated.