Figure 6.
HY5 and HYH Bind to the cis-Regulatory Element T/GHY5-Box.
Biotin-labeled double-stranded probes (40 fmol) were incubated with (+) or without (−) expressed and purified HY5 ([A] and [C]) or HYH ([B] and [D]) protein (400 ng). Binding reactions were resolved on 6% native polyacrylamide gels. WT corresponds to the −117 to −62 fragment of the HY5 promoter, whereas MUT8, MUT10, MUT12, MUT8-10, and MUT8-10-12 carry the corresponding single, double, and triple linker scan mutations in this context. The mE-box probe has mutations identical to those described by Abbas et al. (2014). In (C), reactions were performed as in (A) using HY5 wild-type probe and HY5 protein, but unlabeled HY5 MUT8 or HY5 MUT10 fragments were used as cold competitors at a 200-fold molar excess. In (D), reactions were performed as in (B) using HY5 wild-type probe and HYH protein, but unlabeled HY5 MUT8 or HY5 MUT10 fragments were used as cold competitors at a 200-fold molar excess. Sequences of all probes are provided in Supplemental Table 2. FP indicates free (nonbound) probe. Asterisks mark a nonspecific band that appears independent of the presence of HY5 or HYH protein.