Figure 3.

Electrophoretic Mobility Shift and Competition Assays with c-Myc Recombinant Protein and Allelic Variants of SNP rs2273061 in JAG1

The allele A probe, corresponding to JAG1 intron 3 sequences centering rs2273061 (underlined in the following sequences) was prepared by annealing of the Biotin-labeled oligonucleotide 5′-GACAACCTGTTACCACTTATTTACCTTCTTTA-3′ with the complementary sequence 5′-TAAAGAAGGTAAATAAGTGGTAACAGGTTGTC-3′; the allele G probe, prepared by annealing the Biotin-labeled oligonucleotide 5′-GACAACCTGTTACCACTTGTTTACCTTCTTTA-3′ with the complementary sequences 5′-TAAAGAAGGTAAACAAGTGGTAACAGGTTGTC-3′. Electrophoretic mobility shift assay (EMSA) was conducted with a commercial kit (Panomics, Fremont, CA). 1 ng of Biotin-labeled probe was incubated with 0.36 μg of c-Myc recombinant protein for 30 min at 15°C in a 10 μl reaction volume containing 2 μl 5 × binding buffer and 1 μg poly d(I-C). For competition reactions, the unlabeled probe was used at 660-fold molar excess of the labeled probe. After incubation, the samples were separated by electrophoresis on a 6% nondenaturing polyacrylamide gel with 0.5 × TBE buffer. DNA-protein complexes were electroblotted to Pall Biodyne B nylon membrane (Pall Corp., Pensacola, FL) and visualized by exposure to Chemiluminescent Detection Film (Agfa, Shanghai, China).

Lanes: 1, labeled A probe; 2, labeled A probe + c-Myc; 3, labeled A probe + c-Myc + unlabeled A probe; 4, labeled G probe; 5, labeled G probe + c-Myc; 6, labeled G probe + c-Myc + unlabeled G probe. Binding was not observed with oligonucleotide containing A allele (lane 2) but was present with oligonucleotide containing the G allele (lane 5). Binding to the G allele resulted in a complex that was specifically competed by unlabeled mutant probe containing the G allele (lane 6).