Figure 2.

The 17AA transcriptional activation region functions in the context of the natural WT1 DNA-binding domain. (A) Diagram indicating the regions of WT1 expressed as recombinant His-tagged proteins. The 17AA alternative splice is indicated in black fill. Recombinant proteins were analyzed by SDS-PAGE and Coomassie staining (M, molecular weight markers in kD, are shown at left). (B) In vitro transcription assay using the W₅E4T (containing 5 × WT1 DNA-binding sites) and G₅E4T promoters. Transcripts were detected by primer extension. Transcription levels (quantified by phosphorimager analysis) are presented relative to G₅E4T. (C) In vitro transcription assays were performed and analyzed as for part B except that 200 and 400 ng of each recombinant His-tagged protein was added. A HeLa nuclear extract (NE) or HeLa nuclear extract that had been fractionated over a column containing WT1 DNA-binding sites (Depleted NE) was used as indicated. Transcript levels were quantified by phosphorimager analysis and are presented as fold activation relative to the transcriptional activity of the depleted extract in the absence of a WT1 derivative.