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Figure 5 | BMC Molecular Biology

Figure 5

From: The leukemia associated ETO nuclear repressor gene is regulated by the GATA-1 transcription factor in erythroid/megakaryocytic cells

Figure 5

Detection of DNA-protein interactions using electrophoretic mobility shift/supershift assays in vitro of consensus GATA binding sequences in the 5'promoter of ETO and nuclear extracts from HEL/MEG-01/G1E cells. Sequences for oligonucleotide probes of core consensus and mutated GATA -651, GATA -636 and GATA-619 sites are shown. Arrows marked shift demonstrate primary DNA-nuclear protein interactions; arrows marked supershift demonstrate DNA-nuclear protein-antibody interactions. Results for HEL cells (A-C) are to the left, results for MEG-01 cells (D-F) to the right and results for the G1E (G) is at bottom. For the GATA -636 probe a shift is shown in HEL cells (A2) that is competed for by excess unlabelled probe (competitor) (A3-A5) indicating binding of nuclear extract protein to the biotinylated probe that contains the GATA -636 sequence. In support of this no binding was observed to a probe that contains a mutated GATA -636 sequence (A9). Proteins bound to the GATA -636 probe were "super-shifted" by antibody to GATA-1 (A7) but not with antibody to GATA-2 (A6) indicating specificity of the DNA-protein interaction. Similar results are shown for the GATA -636 probe in MEG-01 cells (D). For GATA -619 and -651 probes a shift is shown (B, C, E, F; lane 2) that is competed for by excess unlabeled probe (competitor) indicating binding of nuclear extract protein to the biotinylated probe that contains GATA -619 or -651 sequences. A supershift is shown with antibody to GATA-1 (B, C, E, F; lane 4) but not with anti-GATA-2 (B, C, E, F; lane 5). To try to distinguish between the binding of GATA-1 and GATA-2 to the ETO promoter EMSA was performed with nuclear extract of G1E cells, which lack GATA-1. MEG-01 nuclear extract was used as positive control (G2). No binding of GATA-2 protein to the consensus ETO promoter was observed (G3) suggesting lack of GATA-2 interaction. These experiments were repeated twice.

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