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. 2007 May 14;204(5):971–977. doi: 10.1084/jem.20052078

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Copyright © 2007, The Rockefeller University Press

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Figure 3. — NF90 regulates ARRE/NFAT and IL-2 transcriptional activation and binds to and regulates IL-2 mRNA stability. (A) Primary cultured thymocytes from NF90^+/+, NF90^+/−, and NF90^−/− littermates were nucleofected with 3× ARRE/NFAT firefly luciferase and EF-1α Renilla luciferase reporter plasmids. Lysates were prepared from nonstimulated (NS) and 6-h PMA/Iono-stimulated (ST) cells and analyzed for the ratio of firefly/Renilla luciferase activities. Data represent the mean ± SD. (B) Jurkat T cells stably expressing NF90 cDNA were established. Control and NF90 T cells were transiently cotransfected with 3× ARRE/NFAT firefly luciferase and EF-1α Renilla luciferase. Lysates prepared from NS and ST cells were analyzed for luciferase activities. *, P < 0.05 for NF90 versus control (n = 4). (C) IL-2 promoter ChIP using mAB 8WG16 to RNA polymerase II. Thymocytes from newborn NF90^+/+ or NF90^−/− mice (two littermate animals for each genotype) were nonstimulated (NS) or stimulated (ST) for 6 h with anti-CD3/CD28, and RNA pol II ChIP was performed. (D) Northwestern analysis. Mouse brain extracts and recombinant NF90 protein were fractionated by SDS-PAGE, transferred to nitrocellulose, and hybridized with ³²P-labeled mouse IL-2 3′ UTR RNA probe (558–939 nt). (E) RT-PCR analysis of IL-2 mRNA induction and stability in NF90^+/+ and NF90^−/− splenocytes nonstimulated (N) or stimulated (S) for 6 h with anti-CD3/CD28. At 6 h, actinomycin D (A) was added, and the remaining IL-2 mRNA after 30 min was determined. (F) Semilog plot of IL-2 mRNA present at 0 and 30 min after addition of actinomycin D. The data shown are the mean and SD of two replicates.