Fig. 3

Biochemical Analyses of the Tob1a and β-Catenin Interaction Unless otherwise stated, the following assays were performed in human HEK293T cells. (A) Interaction between Myc-tob1a and Flag-β-catenin. TCL indicates total cell lysate, which is consistently used hereafter. (B) Colocalization of overexpressed Myc-tob1a (red) and HA-β-catenin (green) in HeLa cells. DAPI was used to identify nuclei. (C) β-catenin/LEF1-stimulated expression of the LEF1-luciferase reporter was inhibited by cotransfection of tob1a plasmid DNA at 100 ng (+), 200 ng (++), or 300 ng (+++). (D) Expression of the LEF1-luciferase reporter in zebrafish embryos was inhibited by tob1a mRNA injection at 150 pg (+), 300 pg (++), or 450 pg (+++), but it was enhanced by injection with 15 ng tob1a-MO. Embryos were injected at the one-cell stage, and luciferase activity was analyzed at the bud stage. (E) Tob1a suppressed association of β-catenin with LEF1. Myc-tob1a doses: 1 μg and 3 μg, respectively. (F) Schematic of different tob1a deletion constructs. NES, nuclear exportation signal; NLS, nuclear localization signal; NRC, nuclear receptor coactivator binding domain; Ub, ubquitination domain. (G) Interactions between Myc-β-catenin and different Tob1a deletion mutants with HA tag. (H) Effect of different Tob1a deletion mutants on β-catenin/LEF1-stimulated expression of the LEF1-luciferase reporter. Note that the mutants ND5, MD2, and MD4, all of which lack the Box B domain, failed to inhibit the reporter activation.