Zic3 binds specifically to its canonical motif.

A, Zic3 consensus motif identified through interrogation of the top 1000 ChIP-seq peaks according to its statistical significance. B, pie-chart showing the frequency of occurrence of the most common binding motif and the Gli motif. C, EMSA with Zic3 canonical motif probe (left panel) and a mutated sequence probe (right panel) demonstrated the specificity of Zic3 binding to its consensus motif. Cy-5 labeled probes containing consensus and mutated motifs were incubated with increasing concentrations (yellow triangle) of Zic3 recombinant protein (Zic3_ZF2-5). A positive shift is indicated by a decrease in mobility (red arrowheads).