Figure 4
AP1 binding affinity to AP1 consensus sequence. Electrophoretic mobility shift assay (EMSA) on HeLa nuclear extract (10 μg). The AP1 consensus oligonucleotide was labelled with γ-32P ATP. Synthetic oligonucleotides, corresponding to the rtMT-A promoter were used as competitors. The constructs used as competitors were: Consensus AP1 (C); AP11,2 (1,2); AP11*,2 (1*,2); AP11,2* (1,2*); AP11*,2* (1*,2*) and AP13,4 (3,4). Mutated AP1 elements are indicated by an asterisk (*). NE, no extract. The location of the AP1 protein complex and free unbound probe is indicated by arrows. (A) Competitor oligonucleotides were added in 400 x molar excess. (B) The AP1 consensus and AP13,4 were added in 200 x and 10,000 x molar excess respectively. (C) Dose–response analysis of AP1 oligonucleotides. Synthetic oligonucleotides, corresponding to the AP1 elements in the rtMT-A promoter were used as competitors. Competitor oligonucleotides were added in 40 x, 400 x and 1000 x molar excess.