Figure 3

Binding of p53 to the MCT-1 promoter region. EMSA was conducted by incubating the MCT-1 promoter probes with nuclear extracts of MCF-10A cells after the genotoxin (ETO) treatment. (A) The biotin-labeled 166 bp probe formed a complex with the nuclear protein (lane 3). This complex was partially depleted by the wild-type (lane 4) but not by the mutant (lane 5) p53-responsive elements in a 100X excess concentration. The presence of p53 in complex was confirmed by inducing a super-shift complex with p53 Ab (lane 6). No specific interaction between the probe and p53 Ab was recognized (lane 1). (B) The biotin-labeled 173 bp probe formed a DNA-protein complex with the nuclear protein (lane 2). This complex was partially reduced by the wild-type competitor at concentrations of 100X excess (lane 3) and 200X excess (lane 4), but it was not depleted by the mutant competitor at a concentration of 100X excess (lane 5). The presence of p53 in complex was confirmed by the p53 Ab inducing a super-mobility shift (lane 6). No specific complex formed between the probe and p53 Ab (right panel). (C) The biotin-labeled 199 bp probe interacted with the nuclear protein (lane 2). This DNA-protein complex was specifically interrupted by wild-type (lane 3) but not the mutant p53-responsive competitor at a concentration of 100X excess (lane 4). A super-shift band was identified as p53 Ab reacted with the DNA-protein complex (lane 5). Again, no particular complex produced among the probe and p53 Ab (right panel).