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Figure 6. Wnt5a promotes nuclear Dvl/Par1b/Smad4 complex formation. (a) Monitoring endogenous Dvl3 and Par1b localization by nuclear-cytoplasmic fractionation of HEK293T cell lysates. The nuclear pool of Dvl3 was enhanced upon transfection of XWnt5a (100 ng/cm2), whereas Par1b is distributed uniformly between the two compartments. LaminB and GAPDH serve as controls for nuclear and cytoplasmic fractionation. (b) Localization of Dvl-GFP (50 ng/cm2) by fluorescence microscopy in HEK293T cells. Note that co-transfection XWnt5a (100 ng/cm2) enhances nuclear localization of Dvl-GFP (green channel). Treatment with the nuclear export inhibitor leptomycinB was carried out at 50 ng/ml for 4 h. Hoechst (blue channel) is a nuclear counterstain. (c) Quantification of cells displaying nuclear localization of Dvl-GFP shown in Figure 6b. Transfected cells were fixed and at least seven fields each containing 10–20 GFP-expressing cells were photographed and counted. LMB dramatically shifts Dvl localization into the nucleus (>95% of cells). Data represent mean and S.D. of two independent experiments. Percentage of nuclear positive Dvl-GFP cells. (d) Endogenous Smad4 interacts with Par1b and Dvl3 from nuclear fractions of HEK293T cells. Wnt5a stimulation increases these nuclear complexes. (e) Endogenous Smad4/Ecto complexes are inhibited in Wnt5a-stimulated HEK293T cells Image published in: Mamidi A et al. (2012) Copyright © 2012 Macmillan Publishers Limited. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives license Larger Image Printer Friendly View

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