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Figure 5


Fig. 5. SoxN represses Wnt signaling in HEK293T cells. (A) Increasing the amount of SoxN decreases the amount of TOPflash activity that is detected. Fold activation is defined as the ratio between relative luciferase units under induced versus uninduced conditions. For comparison between independent experiments, values were normalized to the 0 ng SoxN data point. (B) Schematic diagram of constructs used for Tcf-bindingsite competition assay. FOP-{Delta}L is identical to TOP-{Delta}L in structure but carries mutated Tcf-binding sites. (C) Increasing amounts of TOP-{Delta}L, but not of FOP-{Delta}L, decrease the amount of TOPflash activity that is detected. Fold activation is defined as in A. (D) Extra Tcf-binding sites do not affect SoxN repression of TOPflash activity, expressed as the fold activation with 10 ng pcDNA-SoxNflag/fold activation with 0 ng pcDNA-SoxNflag. Each competitor DNA was present at 2.5x the amount of TOPflash reporter. No statistically significant difference in the degree of repression can be detected. (E) SoxN and Tcf4 at low doses synergistically repress TOPflash activity. Cell cultures containing 10 ng pcDNA-SoxNflag and 0, 5, 25, 50 or 100 ng of pcDNA-Tcf4myc were assayed. Empty pcDNA3.1 vector was used to hold constant the total amount of DNA added. Each bar represents relative activation, defined as fold activation with 10 ng pcDNA-SoxNflag/fold activation with 0 ng pcDNA-SoxNflag. (F) Beta-catenin, but not SoxN, is co-immunoprecipitated with Tcf in TOPflash cell extracts. pcDNA-TCF4myc (0.5 µg) and pcDNA-SoxNflag (0.05 µg) were co-transfected into HEK293T cells and grown under induced and uninduced conditions. Control cultures contained only pcDNA3.1 to reveal non-specific cross-reacting proteins. Cell extracts (e) were subjected to immunoprecipitation using Myc-antibody-conjugated ProtG beads (b). Immunoblot was stained with anti-Flag and anti-beta-catenin. SoxNflag is not detected in the Tcf4-myc-bound fraction under conditions where beta-catenin is found in the induced Tcf4-myc bound fraction (third lane). (G) SoxN and beta-catenin are not co-immunoprecipitated in TOPflash cell extracts. pcDNA-SoxNflag (0.5 µg) was transfected into HEK293T cells and grown under induced conditions. Cell extracts (e) were subjected to immunoprecipitation using flag-antibody-conjugated ProtG beads (b). Immunoblot was stained with anti-flag and anti-beta-catenin. Beta-catenin is not detected in the SoxNflag-bound fraction (first lane).





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