35S De Novo Methylation Figure 5. Gentian nuclear factors bounds to 2149 to 2124 region of the 35S enhancer. (A) Electrophoretic mobility shift assay (EMSA) using 26 bp probes (except 2107 to 283 region with 25 bp) from each of eight different parts of the 35S enhancer region where bisulfite analysis was performed. Each panel shows three lanes (F, free probe; G, probe with gentian nuclear extract; T, probe with tobacco nuclear extract), and indicates each region at the top of the panel. ns, positions of non-specific signals. (B) Determination of binding affinity of gentian or tobacco nuclear extracts with the 2149 to 2124 region probe. The 100-fold molar excess of unlabeled wild-type 2149 to 2124 or 2191 to 2166 region competitors were incubated with the gentian or tobacco nuclear extracts (left panel). Binding reactions in the presence of 2-, 10-, and 30-fold molar excess of the wild-type (wt) or mutated (mt) 2149 to 2124 region competitors were performed in the presence or absence (F) of the gentian nuclear extract (right panel). doi:10.1371/journal.pone.0009670.g005 PLoS ONE | www.plosone.org 7 March 2010 | Volume 5 | Issue 3 | e9670