Fli-1 binds specifically to EBS1 in the SHIP-1 promoter and negatively regulates its expression. (A) Nuclear extracts from CB3 cells were incubated with EBS1-3 sites in the presence or absence of the Fli-1 antibody and nonspecific competitor poly (dI-dC) and subjected to EMSA. Competition assays were performed in the presence of 10-fold excess unlabeled oligonucleotides (cold competitor). (B) Two nucleotides in EBS1 were changed from ACAGGAAGTCA to ACAGGTTGTCA (designated MUT-EBS-1). Nuclear extracts from CB3 cells were incubated in the presence of poly (dI-dC) and γ-³²P-labeled oligonucleotides containing EBS1, MUT-EBS1, or MDM2²⁷  and subjected to EMSA and supershifting with the Fli-1 antibody. Competition assays were performed in the presence of 100-fold excess unlabeled oligonucleotides. (C) Luciferase assays were performed in 293T cells cotransfected with the indicated amounts of a Fli-1-expression vector, either the pGL3-SHIP-1 or pGL3-mut-SHIP-1 vector and the Renilla luciferase vector. The pGL3-SHIP-1 luciferase reporter vector contains a 988-bp region within the SHIP-1 promoter (Figure 4A). Site-directed mutagenesis was used to alter nucleotides ACAGGAAGTCA to ACAGGTTGTCA in EBS1 of pGL3-mut-SHIP-1. The relative luciferase units (RLU) are representative of the firefly luciferase/Renilla luciferase signals (×100). Luciferase assays were performed in triplicates. (D) Fli-1 protein expression in 293T cells transfected with the indicated amounts of the Fli-1 expression vector, relative to HB60-5 and CB3 cells. The 2 Fli-1 protein products are observed as a result of 2 isoforms, 48 and 51 kDa, synthesized by alternative translation initiation through the use of 2 highly conserved in-frame initiation codons.⁴⁷