Fig. 7

Fig. 7

a Cartoon showing the armadillo domain of β-catenin. Arm1 contains armadillo repeats 1–4 and Arm2 contains repeats 5–9. Known core binding armadillo repeats of β-catenin for Axin and APC interaction were indicated with red and green bars, respectively. b IP experiment showing that the Arm1 fragment can interact with ADNP in HEK293T cells. c IP experiment showing that the Arm2 fragment can interact with ADNP in HEK293T cells. d The relationship between ADNP–β-catenin and β-catenin–Axin interactions was examined by performing competitive protein-binding assay. An increasing dose of plasmids encoding FLAG-ADNP and plasmids encoding MYC-AXIN1 and HA-β-catenin was co-transfected into HEK293T cells. Lysates were extracted from the transfected cells and pull-downed by HA antibody followed by WB using MYC and HA antibodies. WB showing that the amount of Axin co-immunoprecipitated with β-catenin became reduced by the increased addition of ADNP. e The competitive protein-binding assay showing that the amount of APC co-immunoprecipitated with β-catenin became reduced by the increased addition of ADNP. f WB showing the expression levels of HA-β-catenin by the addition of Tet-Express protein in the inducible HA-β-catenin Adnp−/− ESCs. g IF staining showing the rescue of TuJ1 levels in day 19 Adnp−/− ESC-derived neuronal cell cultures by adding Tet-Express transactivator daily at early stage of neural induction. h Quantification of mean fluorescence intensity of TuJ1 staining using ImageJ for panel (g), based on three biologically independent experiments (n = 3–5 different regions of interest per group). Data are presented as mean values ± SEM and p values by two-tailed unpaired t-test are shown. i The representative morphology of day 6 neurospheres derived from control, Adnp−/− ESCs, Adnp−/− ESCs treated with CHIR, as well as FLAG-ADNP and HA-β-catenin restoring Adnp−/− ESCs, and the rescue experiments were repeated two times. The WB and IP experiments have been repeated two times and similar results were obtained.