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Consistent with PTD uptake, extracellular PrP binding to the cell surface could be competed with soluble GAGs.
Moreover, the formation of this complex was impaired by pre-incubation with the ER antagonist ICI 182780 and the detection of the complex could be competed with unlabelled TCF-oligonucleotide but not by a mutant TCF oligonucleotide (Figure 7 A).
The complete segregation of short linear vs. long or discontinuous epitopes was entirely unexpected: not a single antibody to the structured region of PrP could be competed with linear dodecameric peptides, whereas all antibodies to the unstructured amino proximal region or to the very far carboxy terminal end of PrP, without any exception, were efficiently competed by cognate peptides.
Furthermore, EMSAs showed that Bin bound specifically to all three of the wild-type Fkh motifs in vitro, and that this binding could be competed with the wild type but not with the mutated binding sites (Fig. 1E), results that correlated with the effect of mutated Fkh sites on enhancer activity.
Moreover, EMSAs showed that CHES-1-like bound specifically to all three of the wild-type Fkh-binding sites in vitro, and that this binding could be competed with the wild type but not with the mutated binding sites (Fig. 1F), results that further strengthen this hypothesis.
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