Figure 4

Direct binding of EMX2 to teneurin-1 promoter oligo-nucleotide probes. (A) Electrophoretic Mobility Shift Assays (EMSA) using EMX2 containing nuclear extract or control nuclear extract were performed in the presence or absence of Dig-labeled probe, unlabeled probe for competition, mutated probes and anti-myc antibody as indicated above the lanes. A specific myc-EMX2/DNA complex could be detected as indicated by an arrow. (B) EMSA with in vitro transcribed and translated EMX2 protein or control extracts were analyzed using Dig-labeled wildtype and mutated probes as indicated. Binding to the probe resulted in a myc-EMX2/DNA complex as indicated by an arrow that was competed by unlabeled probe and resulted in a supershifted complex after addition of anti-myc-antibody as indicated. (C) EMSAs with nuclear extracts of E12.5 embryos and nuclear extract of myc-EMX2 overexpressing cells as a control were performed using Dig-labeled wildtype probe. Binding to the probe resulting in a myc-EMX2/DNA complex and an EMX2/DNA complex is indicated by arrows. (D) ChIP of chicken embryos electroporated with flag-myc-tagged EMX2 (+) and control chicken embryos (-). Fold enrichment of the target region, containing the homeobox binding site versus a negative control region from the coding region of the same gene after anti-flag precipitation is shown. Error bars display standard deviation of the mean.