Assembling the c-Myb/GATA-3 complex on the GATA-3 promoter. (A) A schematic diagram of the GATA-3 promoter region, including the minimal promoter. The region illustrated, nts −148 to +587, contains the start of transcription (arrow) from exon 1b and was used for promoter analysis experiments. Gray circles represent GATA binding sites. The sequences below the promoter scheme show the wild-type and mutated GATA binding sequences. (B) Reporter assays carried out with pG3P-S-GATA-3 wild-type minimal promoter vectors (G3P-GBwt), the reporter constructs with one mutated GATA-3 binding site (G3P-GBmut1 and G3P-GBmut2), or both mutated GATA-3 binding sites (G3P-GBmut1 + 2). The promoter vectors were cotransfected with c-Myb, GATA-3, and/or control expression vectors into to 293T cells and Dual-Luciferase activities were determined as described in the Dual Luciferase Reporter Assay method. (C) Purified nucleoprotein complex was obtained from Jurkat cells 48 hours after transfection of siRNAs with or without pcDNA GATA-3-Flag tag expression vector. ChIP assays were carried out with a GATA-3 antibody for endogenous GATA-3 or with a Flag antibody for exogenous GATA-3. The precipitated DNA fractions were analyzed by quantitative RT-PCR with primers specific to the GATA-3 downstream minimal promoter region. The bands at the bottom of each graph show c-Myb, GATA-3, and β-actin protein expression as determined by Western blot 48 hours after transfection of siRNAs with or without pcDNA GATA-3-Flag tag expression vector. Data are representative of 3 independent experiments.