Figure 4.

Sox2 forms a ternary complex with DNA and RNA. (A) Schematic representation of the RNA fishing experiment to test for the simultaneous binding of RNA and DNA to Sox2. (B) After immobilization of biotinylated DNA on magnetic streptavidin beads and washing, the eluates were analysed by native 10% PAGE gels. Components of the reaction mixtures are labelled on the top of each gel. The lasers used to sequentially detect Cy5-labelled DNA and Cy3-labelled RNA are indicated to the left. Results shown in the same column in upper or lower panels were from identical gels but scanned with different lasers. In: input; FT: flow-through; El: elution. (C) EMSAs of Sox2 constructs with Cy5-DNA and Cy3-RNA. Concentrations of DNA, RNA and protein are labelled with ‘–’, ‘+’ and ‘++’ indicating 0 nM, 50 nM and 100 nM, respectively. In each column, the upper panel and bottom panel are results from identical gels but scanned with the Cy5 (top) or the Cy3 excitation wavelength (bottom). (D) Quantification of the results shown in (C). The barplot shows the mean ± SD (n = 3). (E–H) Competition EMSAs using Sox2-ΔHMG-Cy5-RNA (E), Sox2-HMG-Cy5-DNA (F), Sox2-ΔRBM-RNA (G) and Sox2-ΔRBM-DNA (H) complexes with increasing concentrations of unlabelled Prox1 DNA or unlabelled 12th-24 RNA (from 500 to 4000 nM). Control: free Cy5-Prox1 DNA or Cy5-12th-24 RNA only; ‘-’, Cy5-Prox1 DNA or Cy5-12th-24 RNA with Sox2 constructs but without competitor.