Characterization of the complex formed at the Fra-1 RCE1. Characterization of the complex formed at the Fra-1 RCE1. A. Demonstration of a PI 3-kinase-dependent gel shift with specificity for RCE1. Nuclear extracts (5 μg of protein) were prepared from non-treated (lanes 2, 5, 9), LY294002 treated (lanes 3, 6, 10), or LY294002 treated, washed, and allowed 5 h to recover (lanes 4, 7, 11) PC3 cells. These extracts were then incubated with digoxigenin-labeled double-stranded oligonucleotide probes containing either the wild-type Fra-1 RCE1 (lanes 2–7) or a mutated RCE1 site (lanes 9–11). Additional controls were to perform the incubations in the presence of a 50-fold molar excess of unlabeled RCE1 oligonucleotide (lanes 5–7) and to electrophorese the labeled oligonucleotides in the absence of the nuclear extracts (lanes 1, 8). The reaction mixtures were analyzed by electrophoretic separation on polyacrylamide gels, transfer to membranes and detection with alkaline phosphatase coupled anti-digoxigenin antibodies. The positions of bound oligonucleotide complexes are indicated as C1 and C2, respectively. B. Presence of Sp1 in the complex binding to RCE1. Nuclear extracts were prepared as above from non-treated (lanes 1, 4, 7), LY294002-treated (lanes 2, 5, 8), or LY294002-treated, washed, and allowed 5 h to recover (lanes 3, 6, 9) PC3 cells. Nuclear extracts were either incubated with no antibody, anti-Sp1 antibodies (lanes 4–6), or normal rabbit IgG (lanes 7–9) before the addition of the labeled Fra-1 RCE1 probe. The reaction mixtures were then analyzed as described above and the positions of the oligonucleotide complexes are indicated as C1, C2, and C3. Gunjan Tiwari et al. Mol Cancer Res 2003;1:475-484 ©2003 by American Association for Cancer Research