Xenbase Image ID: 147666

Figure 4. Ectodermin Binds Smad4 (A) Human Ectodermin inhibits TGF-β signaling. The CAGA12 luciferase reporter (100 ng) was transfected in HEK293T cells alone or in combination with human Ectodermin expression plasmid (500 ng) as indicated. After transfection, cells were either left untreated (white bars) or treated with 100 pM TGF-β1 (black bars). Doses are for transfection of 2 cm2 wells. Error bars are standard deviations. (B) Human Ectodermin inhibits BMP signaling. The Vent-2 BMP minimal responsive element (150 ng) was transfected in HepG2 cells alone or in combination with human Ectodermin expression plasmid (500 ng) as indicated. BMP signaling was provided by cotransfected CA-Alk3 and Smad1 expression plasmids. (C) Ectodermin interacts with Smad4 in vivo. Shown is immunoprecipitation (anti-HA antibody) of HEK293T cell lysates transfected with the indicated expression plasmids for different Flag-tagged Smad proteins and HA-tagged Xenopus Ecto. (D and E) A physiological complex between endogenous Ectodermin and Smad4 in HEK293T cells. pep indicates the Ectodermin immunogenic peptide competing for the antibody. (F) In vitro interaction between Ectodermin deletion mutants and Smad4. In vitro-translated 35S-Ecto fragments were incubated with bacterially expressed Smad4 as Sepharose bound GST fusion. Copurifying Ecto fragments were resolved by SDS-PAGE and visualized by autoradiography. The linker region of Ectodermin contains the Smad4 interaction domain. (G) RT-PCR analysis of cultured animal caps (harvested at stage 27) expressing the indicated mRNAs (dose in parenthesis). Lanes 2–4: the neural markers’ inductions (Sox2 and NCAM) by Ecto (400 pg) are downregulated by increasing doses of Smad4 (lane 3, 3 ng; lane 4, 6 ng). (H) In situ hybridization for the mesoderm marker VegT on embryos uninjected (control) or injected in a single blastomere at the four-cell stage with Smad4 mRNA (2.5 ng) alone or in combination with Ecto mRNA (800 pg). Pictures are lateral close-up views, with blastopore lip at the bottom. Dashed line indicates the normal upper limit of VegT expression. Image published in: Dupont S et al. (2005) Copyright © 2005. Image reproduced with permission of the Publisher, Elsevier B. V. Larger Image Printer Friendly View

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