DNA-protein binding at the −39 ETS, CRE, and EGR binding motifs in the human VEGFR1 promoter. (A) EMSA was performed with ³²P-labeled −39 ETS probe in the absence (lanes 1, 9, 12) or presence of nuclear extract from HUVECs (lanes 2-8), recombinant SPI (lanes 10-11), or recombinant ELF-1 (lanes 13-14). In competition assays, a 50-fold molar excess of unlabeled wild-type (lane 3) or mutant (lane 4) −39 ETS probe was added to the reaction mixture. In supershift assays, nuclear extracts or recombinant protein was incubated in the presence of antibodies (indicated by prefix “a”) to ELF-1 (lanes 5, 14), SP1 (lanes 6, 11), ETS-1 (lane 7), or control antibody (lane 8). The arrow indicates specific SP1-DNA complex. NS indicates nonspecific complex. (B) EMSA was performed with ³²P-labeled consensus CRE probe in the absence (lane 1) or presence of nuclear extract from HUVECs (lanes 2-7). In competition assays, a 50-fold molar excess of unlabeled wild-type (lane 3) or mutant (lane 4) CRE probe was added to the reaction mixture. In supershift assays, nuclear extracts were incubated in the presence of antibodies to CREB (lane 5), ATF2 (lane 6), or control antibody (lane 7). indicates specific CREB-DNA complex; *, supershifted complex. (C) EMSA was performed with ³²P-labeled EGR binding site probe in the absence (lane 1) or presence of nuclear extract from HUVECs (lanes 2-5). In supershift assays, nuclear extracts were incubated in the presence of antibodies to EGR-1 (lane 3), EGR-3 (lane 4), or control antibody (lane 5). indicates DNA-protein complex. (D) EMSA was performed with ³²P-labeled EGR binding site probe in the absence (lanes 1-2) or presence of recombinant EGR-1 (lanes 3-8). In competition assays, a 50-fold molar excess of unlabeled wild-type (lane 4) or mutant (lanes 5-6) EGR probe was added to the reaction mixture. In supershift assays, recombinant protein was incubated in the presence of antibodies to EGR-1 (lane 7) or control antibody (lane 8). * indicates supershifted complex. (E) EMSA was performed with ³²P-labeled EGR binding site probe in the absence (lanes 1-2) or presence of recombinant EGR-3 (lanes 3-8). In competition assays, a 50-fold molar excess of unlabeled wild-type (lane 4) or mutant (lanes 5-6) EGR probe was added to the reaction mixture. In supershift assays, recombinant protein was incubated in the presence of antibodies to EGR-3 (lane 7) or control antibody (lane 8). * indicates supershifted complex.