IMAGE

Fig 7

ID
ZDB-IMAGE-200817-18
Source
Figures for He et al., 2020
Image
Figure Caption

Fig 7 Nanog interferes with the binding of β-catenin to Tcf7 in vitro and in vivo.

(A) Nanog interacts with Tcf7 through its N terminal. Different Myc-tagged Nanog were constructed and co-transfection with HA-Tcf7 in HEK293T cells. Among all the mutated types of Nanog, only the N-terminal truncated Nanog (Nanog-ΔN) could not coprecipitate with Tcf7, indicating that Nanog physically interacts with Tcf7 through its N terminal. (B) Tcf7 interacts with Nanog through its GroBD. Different Myc-tagged Tcf7 were constructed and co-transfection with HA-Nanog in HEK293T cells. Among all the mutated types of Tcf7, only the GroBD deleted Tcf7 (Tcf7_ΔGroBD) could not coprecipitate with Nanog, indicating that Tcf7 binds with Nanog through GroBD. (C) Nanog and β-catenin competitively binds with Tcf7. Co-transfection of increasing amount of Nanog decreases the interaction between β-catenin and Tcf7 in a dose-dependent manner. When increased amount of Nanog was transfected into Tcf7 and β-catenin co-transfected cells, decreased amount of β-catenin could be coprecipitated. The molecular weight of HA-Nanog is around 55 Kda, and HA-β-catenin is around 100 KDa, so we could distinguish the 2 anti-HA bands by different protein sizes. (D) Co-transfection of Ctnnbip1 increased the binding affinity between Nanog and Tcf7. Two different amounts of HA-ctnnbip1 were co-transfected with Tcf7, Nanog and β-catenin in HEK293T cells; because Ctnnbip1 was overexpressed, an increased amount of Nanog was coprecipitated by myc-Tcf7. The molecular weight of HA-ctnnbip1 is around 10 KDa. Note that HA-β-catenin level was reduced when Ctnnbip1 was overexpressed. (E) Co-immunoprecipitation assay showed that increased amount of endogenous β-catenin could interact with Tcf7 in MZnanog mutants compared with WT embryos. Embryos were collected at 4 hpf. (F) The intensity ratio of β-catenin to Tcf7 in panel E. Error bars, mean ± SD, *P < 0.05. The P values in this figure were calculated by Student t test. The underlying data in this figure can be found in S1 Data. GroBD, Groucho binding domain; HEK293T cells, human embryonic kidney 293T cells; MZnanog, maternal -zygotic mutant of nanog; hpf, hours post fertilization; WT, wild type.

Figure Data
Acknowledgments
This image is the copyrighted work of the attributed author or publisher, and ZFIN has permission only to display this image to its users. Additional permissions should be obtained from the applicable author or publisher of the image. Full text @ PLoS Biol.