Bipartite Recognition of DNA by TCF/Pangolin Is Remarkably Flexible and Contributes to Transcriptional Responsiveness and Tissue Specificity of Wingless Signaling
Figure 2
Binding preferences of TCF/Pan for various HMG-Helper site configurations in vitro.
(A) Competition electromobility shift assay (EMSA) experiments performed with a recombinant TCF fragment containing the HMG and C-Clamp domains, an AK6 IR-labeled probe, and competitor oligonucleotides containing one of the four orientations at 0 or 6 spaces. Images were taken on the Licor Odyssey, and binding intensity quantified with Image Studio 2.0. AK6 and FF0 were the strongest competitors, but Helper sites in all positions improve binding affinity when compared to binding of the HMG sites alone (HMG only 1 and 2). (B) The IC50 value (the measure of DNA concentration required to reduce binding of the labeled probe to 50% of uncompeted levels) for each competitor was calculated using Prism 6.1 (Graphpad). (C) Semilog graphs depicting competition results from three independent experiments. Error bars indicate SD. Sequences of the HMG and Helper motifs the same as shown in Figure 1D (see Table S1 for full sequence of each competitor).