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. Author manuscript; available in PMC: 2010 Oct 1.
Published in final edited form as: J Immunol. 2009 Oct 1;183(7):4192–4196. doi: 10.4049/jimmunol.0902041

Figure 1. FOXP3 dependent Dab2 expression in regulatory T cells.

Figure 1

A. Semi-quantitative RT-PCR (sqRT-PCR) analysis of Dab2 expression in NFC cells infected with a control retroviral vector (MSCV-Gfp) and a retroviral vector expressing Foxp3 cDNA (MSCV-Foxp3-IRES-Gfp). Csrp2, Cysteine-rich protein 2, is a potential TGFβ-regulated gene that has been classified as a Treg cell signature gene (23). B. Real-time PCR analysis of Dab2 expression in FOXP3+ cells sorted from Foxp3–gfp mice based on GFP expression and from Dab2 CKO mice based on CD25 expression. C. Real-time PCR analysis of Dab2 expression in thymic CD4SP (CD4+CD8–) FOXP3– and CD4SPFOXP3+ cells sorted from Foxp3–gfp mice. Data in B and C are normalized to Actb mRNA expression and are representative of 3 individual experiments. AU: Arbitrary Units D. (left panel) FACS histograms showing FOXP3-GFP expression in sorted CD4+GFP– naïve T cells stimulated with anti-CD3 and anti-CD28 in the presence or absence of TGFβ (2ng/ml) and ATRA (100nM); (right panel) Real-time PCR analysis of Dab2 mRNA expression, normalized to Actb mRNA, in each stimulation condition. Data are representative of 3 individual experiments. AU: Arbitrary Units E. 4 FOXP3 consensus binding sites were identified in the Dab2 gene regulatory element (circles), two upstream of exon 1 (Utr1, Utr2) and two in intron 1 (Int1.1, Int1.2). ChIP assay (one of two independent experiments) on PMA/Ionomycin activated NFC-Foxp3 (F) and NFC-vector alone (V) cells using anti-FOXP3 Ab showed FOXP3 binding only to Utr2 region (filled circle). Anti-FOXP3 antibody binding to Pde3b locus (3) was used as a positive control.