Figure 8.

Determination of NF-κB-binding sequences in the PAcP promoter. (A) ³²P-labeled PSD oligonucleotides and PSD oligonucleotides containing random sequences at the 5′- or 3′-region, or NF-κB consensus oligonucleotides, were incubated with nuclear extracts from PC-3 cells (left panel) or recombinant NF-κB p65 protein (right panel) in the absence or presence of 100 molar excess of unlabeled corresponding probe. (Upper) The authentic PSD oligonucleotide sequence is underlined. (B) Oligonucleotides containing different overlapping sequences from the wild-type PSD oligonucleotide (indicated in bold) surrounded by random sequences were tested for in vitro binding with p65 NF-κB. (C) Effect of point mutation inside the AGGTGT motif on NF-κB-binding. The ³²P-labeled oligonucleotide probes containing the wild-type or mutated AGGTGT motifs surrounded with random sequences were incubated with recombinant p65 NF-κB protein. Mutated oligonucleotides are indicated with italic letters. The nuclear extract was prepared and EMSAs were performed as described under Materials and Methods.