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. Author manuscript; available in PMC: 2012 Dec 1.
Published in final edited form as: J Immunol. 2011 Oct 28;187(11):5974–5982. doi: 10.4049/jimmunol.1100550

Figure 4. c-Myb and GATA-3 bind to CGRE directly.

Figure 4

(A) ChIP assays were performed with anti-c-Myb and anti-GATA-3 antibodies using primary human CD4+ naive and effector/memory T cells after stimulation with IL-4, IL-2 and anti-CD3/CD28 antibodies for 3–4 days. The fold enrichments were measured by QRT-PCR with 8 specific primers upstream of the IL-3 locus including CGRE as shown in the bottom schema. The transcription start site is indicated. (B) Electrophoretic Mobility Shift Assay for binding of c-Myb and GATA-3 to CGRE. Proteins were in vitro translated from their respective pcDNA expression vector using TNT Quick Coupled Transcription/Translation Systems (Promega) and subjected to EMSA with a radiolabeled CGRE oligonucleotide (CGRE) and CGRE oligonucleotide containing Myb's mutant binding sequence (CGRE mut Myb). Oligonucleotide competition was carried out with 10-fold excess of unlabeled CGRE oligonucleotide (CGRE).