Fig. 5

Analysis of p53 bindings on the 13q-Enh enhancer using ChIP assays and EMSA. a The schematic diagram showing the relative positions of six p53 binding sites and five sequences for ChIP and three pairs of EMSA probes to the 5′ boundary of the 13q-Enh. b ChIP assays showed that all the five sequences, but not the negative control sequence S-N, were specifically precipitated with anti-p53 antibodies, indicating p53 binds to the 13q-Enh in vivo. c EMSA assays demonstrated the specific p53 binding to the 13q-Enh and the genetic variations affected the bindings of p53 protein complexes to the enhancer region. Each panel represents the results using the corresponding paired of probes as indicated. Lane 1 and lane 9: negative control using free probes without nuclear extracts; lane 2 and lane 8: using nuclear extract with the biotin-labeled wide-type or mutant probes, respectively; p53 protein complexes formed at wild-type and mutant probes are indicated by black arrows; lane 3: using unlabeled specific wild-type probes pre-incubated with nuclear extracts; lane 4: Sp1 consensus sequence that was used as non-specific control probe; lane 5 and lane 6: using 1 μg and 2 μg anti-p53 antibody to pre-incubate with the nuclear extracts, respectively; lane 7: negative control using 2 μg mouse IgG to pre-incubate with the nuclear extracts