FIG. 4.

Characterization of the nuclear protein that interacts with oligonucleotides containing Sp1 elements and an RCE motif. Nuclear extracts from HeLa cells (2 to 5 μg of protein) were incubated with the ³²P-labeled double-stranded oligonucleotide probes that contained WRN Sp1-3 (positions from −86 to −57) (A), Sp1-2 (positions from −65 to −36) (B), Sp1-1 (positions from −46 to −17) (C), and RCE-1 (positions from −70 to −51) (D) in the absence (lanes 1, 6, 11, and 16) or presence (lanes 2, 7, 12, and 17) of 50-fold molar excess amounts of unlabeled Sp1 oligonucleotides. The mixtures were electrophoresed in a 4% polyacrylamide gel as described in Materials and Methods. Similar experiments were also done with 50-fold excess amounts of unlabeled AP1 oligonucleotide (lanes 3, 8, 13, and 18). In the experiments represented by lanes 4, 9, 14, and 19, the Sp1 protein-specific rabbit IgG antibodies (5 μg) were included in the reaction mixtures before incubation; as the control for these experiments, the same reactions were performed with nonimmunized rabbit IgG fraction (5 μg) (lanes 5, 10, 15, and 20).