The Sox2M203–209mutant does not interact with Grgs and fails to repress a reporter construct. (A) Unlike WT Sox2, the Sox2 M203–209 mutant was unable to translocate Grg5 into the nucleus of co-transfected Cos-7 cells suggesting loss of interaction. Counting the proportion of cells exhibiting altered distribution of MYC-Grg in cells co-expressing Sox2 revealed a highly significant reduction of about 50% (p = 0.0002). (B) Immune precipitation of MYC-Grg5 co-transfected with WT Sox2 or with the Sox2 M203–209 mutant, showing that the mutant failed to be co-precipitated with Grg5. (C) In an in vitro pull down assay, immunoprecipitation of GST-fused Grg1 or Grg5 reproducibly pulled-down 4–5 fold less Sox2 M203–209 than it did WT Sox2 (D) The Sox2 M203–209 mutant retained the ability to activate luciferase expression driven by the 3xSX promoter in Cos-7 cells (the difference between this and the level induced by WT Sox2 was not statistically significant). (E) The ability of co-transfected Sox2 M203–209 mutant to induce luciferase expression driven by the REX promoter in P19 cells was very similar to that induced by WT Sox2. (F) Unlike WT Sox2, the Sox2 M203–209 mutant failed to repress luciferase expression driven from the GFAP promoter, and the addition of Grg3 or Grg5 had no effect on this. *p = <0.05; **p = <0.01; ns – not significant Scale bar approximately 20 μm.