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Figure 3 | BMC Molecular Biology

Figure 3

From: Upstream Stimulating Factors 1 and 2 Enhance Transcription from the Placenta-Specific Promoter 1.1 of the Bovine Cyp19 Gene

Figure 3

The E-box binding factor is composed of USF1 and USF2. A) Western blot analysis of proteins eluted from immobilised target DNA containing a wild type (Ewt), or a mutated E-box motif (Emut). Protein samples were subjected to SDS-PAGE alongside the nuclear extract (NE). Gels were blotted and incubated with commercial anti USF1- and anti USF2-IgG. Both antibodies interacted with nuclear extracts and eluates from the wild type, but not from the mutated target oligonucleotides. B) EMSA experiments with the labelled E-box probe and bovine placenta-derived nuclear extracts. With nuclear extract, a complex (shift) formed (lane 1). Specificity of the complex was demonstrated by competition with a 100-fold excess of the unlabelled probe (lane 2) or a mutated probe (lane 3). Addition of anti USF1 antibodies strongly reduced the complex (lane 4). In the presence of anti USF2 antibodies, a supershift complex formed (lane 5). There is no evidence for more than one specific complex, suggesting that the probe is bound by USF1/2 heterodimers.

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