FIGURE

Fig. 4

ID

ZDB-FIG-210203-4

Publication

Zhang et al., 2020 - VBP1 modulates Wnt/β-catenin signaling by mediating the stability of the transcription factors TCF/LEFs

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Fig. 4

Figure 4. VBP1 interacts with TCF/LEFs and pVHL. A, the interaction between exogenous TCF/LEFs and VBP1. HEK293T cells were cotransfected with FLAG-tagged VBP1 and Myc-Vector, Myc-Tcf7, Myc-Tcf7l1, Myc-Tcf7l2, or Myc-Lef1, respectively. After 48 h, the cell extracts were immunoprecipitated with an anti-Myc antibody. The immunoprecipitates and the inputs were analyzed by Western blotting with indicated antibody. Similar results were obtained from three experiments. B, the interaction among exogenous VBP1, endogenous TCF7L2, and pVHL. HEK293T cells were transfected with FLAG-tagged VBP1. After 48 h, the cell extracts were immunoprecipitated with an anti-FLAG antibody. The immunoprecipitates and the inputs were analyzed by Western blotting with indicated antibody. Similar results were obtained from three experiments. C, VBP1 bound directly to TCF/LEF. GST and GST-VBP1 proteins expressed by bacteria were incubated with GSH beads and the extracts from nontransfected HEK293T cells (for endogenous TCF7L2 detection) or HEK293T cells transfected with Myc-Tcf/Lefs or FLAG-pVHL. Red asterisk indicates the specific band of GST-VBP1. Similar results were obtained from three experiments. D, BiFC assays detected the binding of VBP1 to Tcf7l1/Tcf7l1ΔN/Tcf7l1ΔNLS in HeLa cells. HeLa cells were cotransfected with FLAG-VBP1-VC and Myc-VN-Tcf7l1, Myc-VN-Tcf7l1ΔN, or Myc-VN-Tcf7l1ΔNLS, respectively. After 24h, Venus in green indicates the interaction between VN-tagged and VC-tagged proteins. Tcf7l1/Tcf7l1ΔN/Tcf7l1ΔNLS was detected by an anti-Myc antibody (red). Nucleus was stained with 4′,6-diamidino-2-phenylindole (blue). Scale bar, 10 µm. Similar results were obtained from three experiments. E and F, mapping of the region in TCF7L2 responsible for the VBP1 interaction. Schematic diagram of human TCF7L2 protein domains is shown in (E). Various HA-tagged TCF7L2 deletion mutants were cotransfected with FLAG-tagged VBP1 in HEK293T cells and cell lysates were subjected to co-immunoprecipitation (F). Red asterisk indicates the specific band. Similar results were obtained from three experiments.

Expression Data

Expression Detail

Antibody Labeling

Phenotype Data

Phenotype Detail

Acknowledgments

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