Identification of GATA-1 or GATA-2 binding to the MBP promoter by gel-shift assay. (A) A double-stranded MBP promoter oligonucleotide extending from position −93 to −58 bp was end-labeled with [γ-³²P] ATP and incubated with 1 μg of double-stranded poly(dI-dC) in the presence of 12 μg nuclear proteins from COS 7 cells which were transiently transfected with GATA-1 (lanes 1 and 2) or GATA-2 (lanes 3 and 4) expression vectors, and in the presence of 12 μg nuclear extracts from YJ-11 cells (lanes 5 and 6). A 50-fold molar excess of each unlabeled oligonucleotide (Competitors), identical to the probe in the binding reaction, was added (lanes 2, 4, and 6). The arrow represents GATA-1– or GATA-2–specific bands, which are inhibited by unlabeled oligonucleotides spanning bp −93 to −58 of MBP P2 promoter region. (B) A double-stranded MBP promoter oligonucleotide extending from position −93 to −58 bp was end labeled with [γ-³²P] ATP and incubated with 1 μg of double-stranded poly(dI-dC) in the presence of 8 μg nuclear proteins from COS 7 cells (lane 1), those from COS 7 cells which were transiently transfected with GATA-1 (lanes 2 through 5), or GATA-2 (lanes 6 through 8) expression vectors. The following unlabeled double-stranded competitor oligonucleotides were added at a 100-fold molar excess over the probe oligonucleotide: MBP bp −93 to −58 (lanes 3 and 7) and MBP mutated GATA-consensus site oligonucleotide (lanes 4 and 8). In lane 5, anti–GATA-1 antibody was added to the reaction mixture.