PAI-1 promoter: EMSAs and supershifts of Nur77.

(A) Overexpression of a truncated clone of Nur77 (amino acids 248 to 580 that have full DNA-binding activity) in MCF-7 cells (lanes 1-4) and addition of the E-20 antibody, which recognizes the C-terminus of the Nur77 family of proteins (lane 5). JURKAT cells were induced with ionomycin and PMA (Ion+PMA) and incubated with a radio-labeled NBRE found in the PAI-1 promoter (P, lanes 6-10) and a canonical consensus NBRE (N, lanes 11-14). The extracts were also incubated with E-20 antibody (lanes 8,13). Binding was competed with a 100-fold molar excess of unlabeled oligonucleotides as indicated (PAI-NBRE, P; Con NBRE, N). Specific binding activity of nuclear protein complexes is indicated by black arrows. A supershift resulting from binding of the Nur77 antibody is indicated by white arrows. (B) HUVECs were stimulated with TNFα, and nuclear extracts were incubated with radioactively labeled oligonucleotides containing the NBRE of the PAI-1 promoter (P). The same extracts were incubated with radioactively labeled oligonucleotides containing the NBRE of the PAI-1 promoter that was mutated in the positions indicated in Figure 1D (PAI-T15A, T; PAI-A11C, A). Specific binding activity of nuclear protein complexes is indicated by black arrows.