Effect of dominant-negative ETS domain peptide A on DNA binding and transactivation. (A) Gel mobility shift assay, demonstrating binding of in vitro–translated (IVT) ELF-1 (arrow) to oligonucleotide probe encoding the conserved ETS binding site in the Tie2 promoter (lane 2), compared with control extract (lane 1). The effect of increasing concentrations of peptide A (0.1, 0.5, 0.8, 1.0, 1.1, and 1.2 μm) upon the binding of ELF-1 to the DNA are shown (lanes 3-8). The binding of peptide A to the oligonucleotide probe is shown (arrow). (B) Competition of binding of ELF-1 to oligonucleotide probe with increasing concentrations of a mutant peptide. (C) Competition of binding of Ets-1 and Ets-2 to an Ets-1/Ets-2 consensus oligonucleotide probe with and without 1.2 μm peptide A1 (P), using in vitro–translated Ets-1 (E1), Ets-2 (E2), versus control (C) extracts. (D) Transactivation of the Tie2 promoter by ELF-1. Addition of 0.1- and 1-μM concentrations of peptide A leads to marked inhibition of transactivation. (E) Transactivation of the Flt-1, MCP-1, and urokinase promoters by ETS factors Ets-1 and Ets-2, or the empty mammalian expression plasmid (pCI; Promega, Madison, WI) in the presence or absence of 1 μM peptide A. Results represent mean ± SD. All experiments were performed 3 times in duplicate.