Figure 5

BzpF is a candidate CREB homolog, which binds CRE DNA in vitro. A. The 120-amino-acid region of BzpF, containing the conserved basic-leucine-zipper region is shown (aa 360-480). Amino acids in bold correspond to the CRE-binding motifs in other eukaryotes as shown in alignment with the human CREB (NP_604391, aa 280-318). The underlined region was expressed as a GST-fusion protein in E. coli and used for EMSA. B. The purified BzpF-GST fusion protein was used in EMSA with radioactively labeled oligonucleotides containing the canonical CRE motif (CRE, lanes 1-4) or with a mutated form of CRE (CRE', Lanes 5-7). An autoradiogram is shown. Lanes 1 and 5 - no protein added; lanes 2 and 6 - GST protein alone (without the BzpF fusion); lane 3 and 7 - BzpF-GST fusion protein; lane 4 - BzpF-GST fusion protein with anti-GST antibodies added for super shift. Competition EMSA was done under the same conditions as in Lane 3 but in the presence of increasing amounts of unlabeled CRE' (Lanes 8-10) or CRE (Lanes 11-13) oligonucleotides as indicated above the lanes. The triangles indicate increased ratios of labeled to unlabeled oligonucleotides--1:10, 1:50 and 1:500). Black text indicates the labeled oligonucleotides and grey text indicates the unlabeled competitor.