Figure 4

EMSA assays on NF-1 and Sp1/Sp3 binding sites. A) Competition experiments were performed in the presence of excess unlabelled O-NF1 (lane 4) and unlabelled mutated O-NF1-mut (lane 5). Complexes supershifted by pre-incubation with an NF-1 antibody (NF-1Ab, lane 2) are marked "SS". "NS" indicates non specific binding complexes, the level of which varied between individual nuclear extractions (lanes 1 and 3). "F" indicates free probe. B) Supershift assays using Sp1 (lanes 2 and 5) and Sp3 (lanes 3 and 6) specific antibodies. C) Competition experiments in the absence (lanes 1 and 5) or presence of excess of unlabelled competitors: Lanes 2 and 6, an excess of unlabelled O-Sp1a or O-Sp1b probe respectively. Lanes 3 and 7, an excess of unlabelled mutated oligonucleotide O-Sp1a-mut and O-Sp1b-mut respectively. Lanes 4 and 8, an excess of a consensus Sp1 oligonucleotide (Promega, E323A).