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Figure 4.
Fig. 4. Binding affinity. (a) EMSAs using constructs of the
Tcf3 promoter of different lengths (30mer:
CTTTAAACCTGTTAATGGGAGCGCATTGTG; 26mer:
TTAAACCTGTTAATGGGAGCGCATTG; 22mer: AAACCTGTTAATGGGAGCGCAT;
18mer: ACCTGTTAATGGGAGCGC; 14mer: CTGTTAATGGGAGC; and 10mer:
GTTAATGGGA) at 1 nM were incubated in the presence (+) or
absence (−) of 50 nM mNanHD. The lowest band in each lane
corresponds to single-stranded DNA. (b–g) EMSA with increasing
concentrations of mNanHD (0 to 2 μM) and mutant HD constructs.
Double-stranded cy5 Tcf3 (1 nM) was used in these assays.
Representative results from at least three independent
experiments are shown. Apparent dissociation constants were
estimated by plotting the fraction of bound protein averaged
over three to five independent experiments against the total
protein concentration followed by fitting a single-site
saturation curve using SigmaPlot. The indicated error represents
the standard error of the fit.
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