Skip to main content
. 2009 Nov 9;30(2):372–381. doi: 10.1128/MCB.00063-09

FIG. 4.

FIG. 4.

The C-terminal domain of KLF4 and the DNA-binding domain of TCF4 interact. (A) HEK293T cells were transfected with Flag-β-catenin, Flag-KLF4, or both, and after 48 h, lysate was collected and incubated with GST-tagged TCF4(1-65). After several washings, bound proteins were eluted and an anti-Flag Western blot assay was performed. (B) HEK293T cells were transfected with Flag-tagged KLF4, with or without HA-tagged TCF4, lysate was harvested, and anti-HA immunoprecipitation was performed. An anti-Flag Western blot assay was then performed on the bound proteins. (C) GST-TCF4(1-65) or GST-TCF4(265-496) was incubated with lysate from HEK293T cells overexpressing various Flag-tagged KLF4 constructs as shown. After several washings, bound proteins were eluted and an anti-Flag Western blot assay was performed. (D) HEK293T cells were transfected with a Myc-tagged TCF4 construct. Forty-eight hours later, cell lysate was collected and incubated with various GST-tagged KLF4 constructs. After several washings, bound proteins were eluted and an anti-Myc Western blot assay was performed. Coomassie staining was performed to demonstrate expression levels of the various GST-tagged proteins used. (E) KLF4ΔM1 binds both β-catenin and TCF4. Left: the Myc-tagged β-catenin C terminus was cotransfected with Flag-tagged KLF4ΔM and KLF4ΔM1 into HEK293T cells. β-Catenin was immunoprecipitated with an anti-Myc antibody, and KLF4ΔM and KLF4ΔM1 were analyzed by Western blotting with an anti-Flag antibody. Right: Flag-tagged KLF4ΔM and KLF4ΔM1 were transfected into HEK293T cells and then incubated with GST-TCF4(265-496). TCF4 was pulled down with GST beads, and KLF4ΔM and KLF4ΔM1 were analyzed by Western blotting. (F) KLF4 directly interacts with TCF4. GST-tagged TCF4(1-65) and TCF4(265-496) were incubated with MBP-tagged KLF4 or β-catenin. TCF4 proteins were pulled down with GST beads, and KLF4 and β-catenin were analyzed by Western blotting with an anti-MBP antibody. Coomassie staining demonstrates the expression level of each purified protein (lower panel).