Predicted and observed transcription factor binding to the CRTh2 proximal promoter.

<p><b>(A)</b> The <i>CRTh2</i> proximal promoter contains putative NFAT and GATA binding sites, which show varying degrees of evolutionary conservation. <b>(B)</b> Site-directed mutagenesis of the CRTh2pro reporter vector to delete the GATA/NFAT/GATA containing-region. <b>(C)</b> Specific deletion of the conserved (-125/-94) base pairs reduced both overall luciferase activity of the vector and response to activation with PMA/Ionomycin. <b>(D)</b> This region was used as a probe for EMSA. Nuclear extracts were made from <i>in vitro</i> differentiated CRTh2⁺ Th2 cells following the resting phase (IL-2) and were re-stimulated <b>(3h PI)</b>. NFAT1 and GATA3 bound to the <i>CRTh2</i> promoter, although NFAT1 binding is more prominent after PMA/ionomycin stimulation Competitors and super-shifting antibodies used are noted above the corresponding lanes. Antibodies denoted as α. Gels are representative of n = 8 from 6 independent cell lines. *p< 0.05, determined by Student’s t-test.