Fig. 8

Electrophoretic mobility shift detected the DNA-binding ability of Sp1 on the ERCC6 promoter. The DNA-protein complex (indicated by black arrow) can be observed (lanes 3, 4, 5, 7, and 9). In contrast, no nucleoprotein complex was observed when used the M-probe and labeled mutated type probe (lanes 1 and 8). The competition assay was done by the addition of 50-, 100-, or 200-fold molar excess of unlabeled methylated probes to the incubation mixtures (lanes 4, 5, and 6). The complex formation was fully suppressed by the addition of a 200-fold molar excess of unlabeled wild type probe (lane 6) and not was suppressed by 200-fold molar excess of unlabeled mutated type probe (lane 9). A supershift band (indicated by black arrowhead, lane 7) was detected when anti-Sp1 antibody was incubated