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EMSA confirms direct binding by the Pax6 paired domain to an evolutionary conserved binding site in the mouse Dkk3 promoter, and a mutated version of this site is not longer able to mediate transcriptional activation by Pax6 from the Dkk3 promoter.

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posted on 16.07.2014, 03:24 authored by Siri Forsdahl, Yury Kiselev, Rune Hogseth, Janne E. Mjelle, Ingvild Mikkola

A, affinity purified recombinant GST-fusion proteins containing the Pax6- or the Pax6(5a) paired domain (PD) were used in EMSA with a 55 bp probe covering the evolutionary conserved BSAP (Pax5) binding site in position −56/−24 at the mouse Dkk3 promoter. A mutated version of this probe is also included. The “5aCON” WT probe (position +180/+208 according to TSS-2), optimized (OPT) and mutated (MUT) versions of this probe were also used in binding reactions with recombinant Pax6 PD and Pax6(5a) PD. The sequences of the wild type (WT) and mutated (MUT) “BSAP” probes are shown beneath the gel. The exact localization of the three bioinformatically predicted- and overlapping BSAP sites is indicated by three lines above the “BSAP” probe sequence. The nucleotides changed in the “BSAP” MUT sequence are underlined. “5aCON” OPT is an optimized version, similar to the original 5aCON sequence. Grey bars show the homology with 5aCON for each version of the mouse Dkk3 “5aCON” probes. B, transient transfections and reporter gene assays in HeLa cells with pGL3-mDkk3 promoter construct nr 2 (−193/+219) containing the mutated “BSAP” and “5aCON” sites, and a deleted “5aCON” site (“5aCON Δ”). The ten deleted nucleotides in the “5aCON Δ” probe which is replaced with the dinucleotide TT are indicated by a Δ symbol above the sequence in A. C, transient transfections and reporter gene assays in HeLa cells with the minimal 184 bp pGL3-mDkk3 promoter construct, nr 6 (−193/−9) containing the mutated “BSAP” site. Transfections were done as described in figure 3.

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