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Fig. 3

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ZDB-IMAGE-070823-39
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Figures for Xiong et al., 2006
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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.

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Reprinted from Developmental Cell, 11(2), Xiong, B., Rui, Y., Zhang, M., Shi, K., Jia, S., Tian, T., Yin, K., Huang, H., Lin, S., Zhao, X., Chen, Y., Chen, Y.G., Lin, S.C., and Meng, A., Tob1 controls dorsal development of zebrafish embryos by antagonizing maternal beta-catenin transcriptional activity, 225-238, Copyright (2006) with permission from Elsevier. Full text @ Dev. Cell