Fig. 1

The Ephb4-2 venous enhancer contains functional ETS motifs. A. ClustalW alignment of the human and mouse sequences of the Ephb4-2 enhancer annotated with conserved ETS binding motifs (green), SMAD4 binding motifs (light blue) and SMAD1/5 binding motifs (dark blue) as previously reported (Neal et al., 2019). Flanking regions outside core ETS binding motifs which adhere to the ERG consensus motifs are indicated in yellow. ∗ denotes nt conserved between human and mouse sequences. B-E. Radiolabelled oligonucleotide probe encompassing a known ETS binding motif (ETS control consensus binding site, B and D) or putative Ephb4-2 ETS motif (ETS-b, ETS-c, ETS-e, ETS-h and ETS-j, C and E) was incubated with either unprogrammed TNT lysate (un), recombinant ETS1 DNA binding domain protein (ETS1-DBD, B–C) or ERG protein (D–E). Competitors added were either water control (−), an excess of unlabelled self-probe (ETS control site) or a single putative Ephb4-2 ETS wildtype (WT) or mutant (MU) motif. Gel shifts denoting protein binding are indicated by green (ETS-DBD) and yellow (ERG) arrowheads, unlabelled probe is indicated by black arrowhead.