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. 2017 Jul 26;8(41):70214–70225. doi: 10.18632/oncotarget.19603

Figure 5. The TGIF1 homeodomain is essential for promoting Wnt signaling.

Figure 5

(A) Nuclear and cytoplasmic fractions were separated from LoVo cells and subjected to immunoblotting with the indicated antibodies. (B) Diagrams showing TGIF1 domain deletion constructs. FL for full length; dHD for homeodomain deletion and dRD-2A for RD-2A domain deletion. (C) Wild-type TGIF1 and various deletion constructs were co-transfected with Topflash-luciferase into HEK293T cells. After 24 hours, the cells were treated with Wnt3a conditional medium for another 24 hours and then harvested to measure luciferase activity. ***, p < 0.001; ns, p>0.05. (D-E) HEK293T cells were transfected with Topflash-luciferase together with TGIF1, AXIN1, wild-type β-catenin or SA-β-catenin as indicated. At 48 hour post-transfection, the cells were harvested for luciferase assay. *, p <0.05; ns, p>0.05. (F-G) HEK293FT cells were co-transfected with constructs carrying β-catenin, TGIF1 and dHD-TGIF1 as indicated for 48h before they were harvested. Cell lyses were precipitated with anti-Flag antibody, followed by anti-Myc immunoblotting (IB) to detect the interaction between β-catenin and TGIF1. IP, immunoprecipitation; WCL, whole cell lysate. (H) HEK293FT cells were co-transfected with constructs carrying β-catenin, TGIF1 and TCF4 as indicated for 48h before they were harvested. Cell lyses were precipitated with anti-HA antibody, followed by anti-Myc immunoblotting (IB) to detect the interaction between β-catenin and TCF4.