Figure 4

Electrophoretic mobility shift assays for SNP-3608T>C with nuclear extracts prepared from RIN-1027-B2 cells. Four probes used for EMSA contained respectively -3608T-3607C, -3608C-3607C, -3608T-3607T and -3608C-3607T. Specific complex formations (compared lines 2 and 5) are indicated by two arrows. Lane 1 is radiolabeled probes without nuclear extract. In the presence of the -3608T allele and the -3607C allele, two bands (bands 1 and 2) were observed corresponding to the fixation of two different factors (lane 2). The intensity of the band 2 decreased when either of these two alleles was changed (lanes 6, 9 and 12 compared to lane 2). The band 2 decreased by 1.5 fold in a TT or a CT configuration (lanes 9 and 12) and completely disappeared in a CC configuration at both loci (lane 6). The intensity of the band 1 and 2 was decreased when non radioactive competitors (either C or T alleles at the -3608 locus) were added to the reaction (lanes 3 and 4 compared to lane 2). In the TT configuration at the two loci, the addition of the -3608C non labeled probe did not decrease the band 2 signal unlike the -3608T probe (lanes 10 and 11 compared to lane 9). These observations suggest that the band 2 corresponds to the binding of a putative nuclear protein when both the -3608T and the -3607C alleles are present. The band 1 was observed when at least one C allele was present at either locus, decreased by 2 fold in a TT configuration (lanes 2, 6, 12 compared to lane 9).