Figure 5

NFAT-binding genes being Itk- and calcineurin-dependent. a. Promoter regions with NFAT-binding sites in IL2, IL7R, Bub1, Slfn1 (Schlafen1), Ctla2a and Ctla2b genes are shown with the binding site(s) represented by black boxes. This identification was done by bioinformatic analyses of the 500 bp region upstream of each gene's transcriptional start site. This approach identified 1–2 NFAT binding sites within the promoter regions of these genes as well as upstream of the translation initiation of Slfn1 gene. The numbers below each box represent the position of the binding site in correlation to the transcription start. The arrows indicate forward and reverse primers. b. NFATc1-binding in IL2, IL7R, Bub1, Slfn1, Ctla2a and Ctla2b genes. CD3⁺ T-cells were isolated from Wt thymus (thy) and spleen (spl) as described in experimental procedures. The PCR pictures were analyzed with Fluoro-S gel documentation equipment (BioRad Laboratories, CA) with a CCD camera, and further evaluated using the Quantity One software. Input; DNA before IP, NTC; no template control. c. Signal intensities of the six genes in Wt unstimulated, Wt anti-CD3-stimulated, Wt anti-CD3-stimulated + CsA-treated, Itk-defective unstimulated and Itk-defective anti-CD3- stimulated samples. The figure was made in dChip [21]. The color scale in the lower part of the picture corresponds to the mean expression of a gene. The red color represents expression level above mean expression of a gene across all samples, the white color is mean expression and the blue color represents expression lower than the mean.