Figure 3.

NFX1-91 binds to the proximal putative X box in the hTERT promoter. EMSA assays. (A) His-tagged recombinant NFX1 protein fragments, schematically represented at the top of the figure, were generated and purified from E. coli. Titrations of His-tagged NFX1-91 (His-N91) were able to shift a wild-type (WT) probe of 48 bp surrounding the proximal E box and including the overlapping putative X box. This shift was slightly diminished on mutation of five of the nucleotides within the putative X-box region (MUT). His-tagged NFX1-123 (His-N123) was much less efficient at binding and shifting the same probes. (B) A C-terminal peptide of NFX1-91 was sufficient to induce a small shifting (brackets) of the wild-type (WT) X-box DNA probe that could be dramatically supershifted (arrowhead) with titrations of a rabbit polyclonal antibody (Ab) raised to this peptide. The last three lanes demonstrate that antibody alone is unable to induce a similar shift.