Journal: Developmental biology

Article Title: Direct activation by Ets and Zic is required for initial expression of the Brachyury gene in the ascidian notochord.

doi: 10.1016/j.ydbio.2007.03.034

Figure Lengend Snippet: Fig. 4. Hr-Ets protein binds to the Hr-Bra promoter. (A) Schematic drawing of Hr-Ets. The black box indicates the Hr-Ets DNA-binding domain (DBD). A purified recombinant DBD protein was used in the binding assay. A pointed domain, which is located near the MAPK phosphorylation site and binds to MAPK in other organisms (Brunner et al., 1994; Wasylyk et al., 1997), is conserved in Hr-Ets. (B) Probe sequences used in binding assay (Fig. 1, red underlines). Core binding sequences for Ets (GGAA/T or complementary) are shown in red. Underlined lower case letters indicate the mutated nucleotides. (C) Electrophoretic mobility shift assay. Arrowhead indicates shifted bands (lanes 1, 4, 7). The binding activities were absent with either mutant probes (lanes 2, 5, 8) or 100× excess unlabeled competitor (lanes 3, 6, 9). No binding activity was detected with other probes (lanes 10–15).

Article Snippet: After removal of the template DNA by digestion with DpnI and phosphorylation with T4 polynucleotide kinase (Toyobo), the amplified fragment was self-ligated.

Techniques: Binding Assay, Purification, Recombinant, Phospho-proteomics, Electrophoretic Mobility Shift Assay, Mutagenesis, Activity Assay