Figure 3.

EMSA analysis of the ets binding site on the CD5 promoter. The ³²P-labeled oligonucleotide probe containing the Ets binding site (5' CGC GTA CAG GGA GGA AGT TGA CCT CGA 3') on the CD5 promoter was incubated with nuclear extracts prepared from M12 cells. The Ets-1 protein:DNA complex is indicated as complex 1. Lane 1 is the probe alone; lane 2, probe plus M12 nuclear extract from transfection with pEVRF0; and lane 3-9: probe plus M12 nuclear extract from transfection pEVRF-ets-1. Competitive EMSA was performed using molar excess unlabeled olionucleotides as indicated: in lane 4, ten-fold molar excess of unlabeled homologous Ets oligonucleotides; Lane 5, 100-fold molar excess of unlabeled Ets oligonucleotides; Lane 6, ten-fold molar excess of unlabeled κE2; and lane7, 100-fold molar excess of unlabeled κE2 oligo. Inhibition EMSA was also performed using antibodies as indicated: lane 8, anti-Ets-1 antibody; and lane 9, pre-immune antibody. F indicates the migration of the free probe.