Binding of Egr-1 to the −32 to −7 flt-1promoter sequence after endothelial denudation or PMA treatment.
(A) Electrophoretic mobility shift assays were performed with 3 μg nuclear extracts from HUVEC incubated with a 32P-labeled DNA probe of the (−32 to −7) flt-1 promoter region. Cell monolayers were mechanically denuded or treated with PMA (50 ng/mL) for the indicated times. C, control untreated cells. The arrow indicates the inducible complex. (B) Supershift assays were performed with polyclonal specific antibodies against Egr-1 and Sp-1 transcription factors using the same probe as in A. Ab, antibody; denud., mechanical denudation. The arrows indicate the supershifted complexes. These results are highly reproducible in more than 3 independent experiments.
