GATA-1 is directly involved in EPO-induced downregulation of EPOR mRNA level. (A) Specific recognition of GATA-protein to the oligonucleotide probe containing GATA-binding consensus sequence (A/T)GATA(A/G) located at the EPOR promoter region. Nuclear extracts from UT-7 cells were incubated with labeled wild-type oligonucleotide probe, and EMSA was performed as described in the Materials and Methods. Unlabeled wild-type or mutant oligonucleotides were added at an excess of the indicated fold. (B) Specific binding of GATA-1 protein to GATA-binding consensus sequence located at the EPOR promoter region. Nuclear extracts prepared from UT-7 cells were preincubated with an excess of antimurine GATA-1 polyclonal antibody, antichicken GATA-2 monoclonal antibody, or preimmune serum, then incubated with a double-stranded ³²P-labeled oligonucleotide probe containing the GATA-binding consensus sequence or the mutant oligonucleotide probe, and EMSA was performed as described in the Materials and Methods. The GATA-1 protein extracted from MEL cells was used as a positive control and supershifted by preincubation with antimurine GATA-1 monoclonal antibody (*). Supershifted complexes are indicated (SS). (C) Time course of specific GATA-1–binding activity detected in EPO-stimulated UT-7 cells. GATA-1–binding activity induced by EPO treatment was analyzed by EMSA method. A double-stranded ³²P-labeled wild-type oligonucleotide probe was incubated with nuclear extracts from the UT-7 cells stimulated with 10 U EPO/mL for the indicated times. Unlabeled competitor DNA was added at 150-fold excess. EMSA with the oligonucleotides corresponding to Sp1 consensus sequence (upper strand: 5′-ATTCGATCGGGGCGGGGCGAG-3′ ) was simultaneously performed to show the amount of protein loaded on the gel.