Fig. 7

Tob Proteins from Different Species Have Conserved Activity (A) Endogenous β-catenin was associated with TOB1 in human HEK293T cells. Mouse IgG was used as control. (B) Interactions between Flag-tagged β-catenin and different Tob proteins with a Myc tag in HEK293T cells. (C) β-catenin/LEF1-stimulated expression of the LEF1-luciferase reporter was attenuated by coexpression of individual Tob constructs. Data were averaged from three independent experiments and are expressed as means plus standard deviations. (D) Endogenous SMAD3 was associated with TOB1 in human HEK293T cells. Rabbit IgG was used as control. The band representing SMAD3 is indicated by an arrow. (E) Interactions between Flag-tagged Smad3 and different Tob proteins with a Myc tag in HEK293T cells. (F) Smad3-induced expression of the CAGA₁₂-luciferase reporter in Mv1lu cells was suppressed by coexpression of individual Tob constructs. Data were averaged from three independent experiments and are expressed as means plus standard deviations. (G) Embryos injection with 400 pg Tob1, Tob2, or tob1b mRNA showed a decrease of chordin and gsc expression at the shield stage and of otx2 expression at the bud stage, while expression of bmp2b at the shield stage and gata1 at 24 hpf was expanded. Embryos are shown in animal pole views for chordin, in anterodorsal views for otx2, in dorsal views for gsc, and in lateral views for bmp2b and gata1. Corresponding statistical data are shown in Figure S10. (H) Models for antagonizing effects of Tob1 on the transcriptional activities of β-catenin and Smad3. Tob1 binds to β-catenin and precludes β-catenin from binding the coactivator LEF1 (left). Tob1 can also bind to Smad3 and prevents the formation of the Smad3/p300 transcriptional complex (right). BTC, basal transcription complex.