A r t i c l e downstream relative to the GATA3 translational start site), and on this basis corresponding primer pairs (S1–S11) were prepared (Fig. 3 A). Splenic CD4 T cells were stimulated with IL-4 for 1 or 24 h, after which these stimulated cells were subjected to a ChIP assay with an anti-STAT6 antibody. Apparent binding was detected by the S4 and S7 primer pairs (Fig. 3 B). STAT6 binding was observed 1 h after stim- ulation and maintained for at least 24 h. A ChIP assay and quantitative PCR using CD4 T cells cultured under Th2 conditions for 1 h provided quantitative confirmation of the Figure 3. Identification of the binding site of STAT6 at the GATA3 gene locus. (A) Schematic illustration of the GATA3 gene locus. 11 putative STAT-binding sites designated as S1–S11 (black dots) are indicated. The blue panel shows the conservation track obtained from the University of Califor- nia, Santa Cruz Genome Browser. (B) CD4 T cells were stimulated with 100 U/ml IL-4 and immobilized anti-TCR mAb for the indicated periods. The stimu- lated cells were cross-linked with paraformaldehyde and then sonicated. The lysates were subjected to a ChIP assay with anti-STAT6 mAb or control Ig. The results are representative of three independent experiments. (C) CD4 T cells stimulated in medium or under Th2 cell culture conditions for 1 h were subjected to a ChIP assay. The levels of STAT6 binding at the S4 and S7 sites were determined by a quantitative PCR analysis. Three independent experi- ments were performed with similar results. The relative intensity (mean of three samples) is shown with SDs. (D) EMSA was performed using radiolabeled double-strand probes containing S4, Mut-S4, S7, or Mut-S7 and nuclear extracts from IL-4–stimulated WT or STAT6-deficient Th2 cells. Results are repre- sentative of two independent experiments. (E) EMSA with nuclear extracts from WT Th2 cells and radiolabeled S4 or S7 probes. Antibodies against p300, CBP, STAT6, or control mouse Ig were added to the reaction to supershift the STAT6–DNA complex. The results are representative of two independent experiments. JEM VOL. 207, October 25, 2010 2497