FIG. 3.

EMSA of GATA-1 binding to the GATA motif at nt −580 to −556 of the HBG2 promoter. (A) Nuclear extracts from K562 cells (lanes 2 to 5) or MEL cells (lanes 7 to 10) were incubated with a ³²P-end-labeled double-stranded oligonucleotide probe corresponding to the wild-type GATA motif. Competition experiments were carried out in the absence (lanes 2 and 7) or presence of a 100-fold molar excess of unlabeled wild-type GATA (lanes 3 and 8) or mutant GAGA (lanes 4 and 9) probe. Lanes 1 and 6 contained no nuclear protein in the incubation. The position of the DNA-GATA-1 complex is marked by an arrow. Note the supershifted DNA-GATA-1 bands (lanes 5 and 10) due to the addition of anti-GATA-1 antibody. Lanes 11 and 12, reference GATA probe (5′-CGCCGCAGAGATAAGGCACTGCC-3′) obtained from Active Motif (Carlsbad, CA) was incubated with K562 nuclear extract, with or without anti-GATA-1 antibody. (B) Competition assays were performed with increasing amounts of non-³²P-labeled wild-type probe at 100-, 50-, and 25-fold molar excess (lanes 2 to 5 for K562 and lanes 6 to 9 for MEL nuclear protein). (C) ELISA-based GATA competition assays. The x axis shows the concentration of the competitor oligonucleotides added. The y axis shows the amount of GATA-1 bound, assessed by colorimetric readings at an optical density of 450 nm (see the text). This is one representative experiment of three similar experiments.