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Binding of Elf5 to regulatory regions of the CCND2 gene. A. A schematic map of the cyclinD2 gene showing the genomic structure and the location of the Elf5-ChIPed segment in the 5' upstream region (circle). The primer sets used for ChIP experiments are shown. B. Gelshift experiments to demonstrate binding of recombinant Elf5 to cyclinD2 regulatory regions. The Elf5-DNA complex is supershifted (*) with the addition of two different antibodies against Elf5, Ab1 (N-20) and Ab2 (generated in the laboratory). Lanes 1-7 represent data with the oligonucleotide corresponding to the cyclinD2 promoter, whereas lanes 8-12 and 13-17 correspond to two segments of the upstream region that contain Elf5-binding sites. C. In vivo occupancy of Elf5 on mouse Ccnd2 gene. ChIP was performed on mouse mammary glands using antibodies recognizing Elf5 as well as a nonspecific IgG as indicated. Input represents PCR amplification of 1% of the genomic DNA. Primer pairs P1-P2 and P3-4 correspond to the upstream and the promoter region respectively. P5-P6 corresponds to 3' region of Ccnd2 gene and serves as a negative control. Results shown are a representative of two independent experiments. D. The cyclin D2 promoter is repressed by Elf5 in reporter gene assays in mammary epithelial cells. The wildtype (black) or mutant (white) cyclinD2-Luc plasmid was co-transfected with expression plasmids encoding Elf5 or Elf3 into MCF-7 cells. Luciferase values were determined and normalized against β-galactosidase values. The corrected luciferase values for cells transfected with empty vector pCMV-HA were set at 1.