ELF-1 and SP1 bind to the −52 ETS motif in the human VEGFR1 promoter and induce promoter activity. (A) EMSA was performed with ³²P-labeled −52 ETS probe in the absence (lane 1) or presence of nuclear extract from HUVECs (lanes 2-7) In competition assays, a 10-fold or 50-fold molar excess of unlabeled wild-type (lanes 3-4) or mutant (lanes 5-6) −52 ETS probe was added to the reaction mixture. indicates specific DNA-protein complexes. (Based on EMSA in panel B, complexes a and c represent SP1 and ELF-1, respectively; the identity of complexes b and d is unknown). NS indicates nonspecific complex. (B) EMSA was performed with ³²P-labeled −52 ETS probe in the absence (lanes 1, 8, 11) or presence of nuclear extract from HUVECs (lanes 2-7), recombinant SPI (lanes 9-10), or recombinant ELF-1 (lanes 12-13). In supershift assays, nuclear extracts or recombinant protein was incubated in the presence of antibodies (indicated by prefix “a”) to ELF-1 (lane 3, 13), SP1 (lane 4, 10), ETS-1 (lane 5), ETS-2 (lane 6), or control antibody (lane 7). indicates SP1 and ELF-1 DNA-protein complexes. (C) EMSA was performed with ³²P-labeled consensus ETS-1 binding probe (lanes 1-3) or −52 ETS (lanes 4-5) in the absence (lanes 1, 4) or presence of in vitro translated ETS-1 ± antibodies to ETS-1 (lane 3). (D) HUVECs were transfected with control siRNA or siRNA against ELF-1 and/or ETS-1. Real-time PCR was used to assay for mRNA expression of ELF-1, ETS-1, or VEGFR1. The results show the means and SDs of mRNA expression (relative to control siRNA-transfected cells) obtained in triplicate from 3 independent experiments. RQ indicates relative quantitation. *P < .05; **P < .01; ***P < .001, relative to control siRNA. (E) ChIP assay was performed using HUVECs. DNA was sheared and resulting DNA-protein complexes were immunoprecipitated in the absence or presence of antibodies to ELF-1, SP1, or control IgG. Real-time PCR analysis was performed using the precipitated DNA fragments and primers for VEGFR1 proximal region, which included the −52 ETS site. (F) Cotransfection assay was carried out in HEK293 cells using 0.3 μg of pcDNA3-SP1 expression vector or empty vector (PCI-pcDNA), and either wild-type VEGFR1-luc (WT) or a similar construct containing a mutation of the −52 ETS site. Data represent mean ± SE of 6 replicates. Luciferase light units are expressed as fold induction over the empty expression vector. (G) Cotransfection assay was carried out in HUVECs using 0.3 μg of pcDNA3-SP1 expression vector or empty vector (PCI-pcDNA), and either wild-type VEGFR1-luc (WT) or a similar construct containing a mutation of the −52 ETS site. Data represent mean ± SE of 6 replicates. Luciferase light units are expressed as fold induction over the empty expression vector. (F-G) *P < .05; **P < .01; ***P < .001, relative to pCI + pcDNA.