Figure 5

The Sox2^M203–209mutant 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.