EMSA illustrating allelic DNA-protein interactions in the promoter region of CDKN2B. Labeled double-stranded oligonucleotide (ds-oligo) probes corresponding to the CDKN2B −287C>G alleles were incubated with HeLa nuclear extracts. Lanes 1 to 3 represent labeled −287G ds-oligos; lanes 4 to 6, labeled −287C ds-oligos. The unlabeled probes used to compete DNA-protein interactions (in 50-fold molar excess) are indicated (+) at the top of each lane. Probe sequences are listed in Table 1. Fast migrating unbound probes can be seen at the bottom of the gel (NS indicates nonspecific), and the position of the DNA-protein complexes of slower mobility are marked by arrows. In this experiment, 3 distinct complexes were found following incubation of the probes with HeLa nuclear extract. Complex 1 was observed with both labeled ds-oligos −287G and −287C (lanes 1 and 4) but was competed by both unlabeled probes, indicating unstable DNA-protein interactions. Complex 2 was also found with both alleles; the −287C-derived complex was competed by both unlabeled probes; however, the −287G-derived complex seemed to be less affected by competitors and thus more stable, suggesting higher binding affinity. Complex 3 appeared only when −287C was present (lane 4 vs lane 1). The specificity of this interaction was illustrated through competition with the specific unlabeled probe, which did not occur with the mismatched −287G probe (lane 6 vs lane 5).