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Figure S3 (A) CoIP of Smad4 in a complex with Par1b. Both proteins were overexpressed. See Fig.3C for association at physiological levels. (B) Luciferase assay for TGFβ in MDA-MB231 cells. The overexpression effects of wild-type Dvl3 are compared with those of two Dvl3 mutants: one that is unable to bind Par1b (5), and one that carries Serine-to-Alanine mutation of the two main Par1b phosphorylation sites (6). While the Par1b-binding domain is required for the effects of Dvl3, the phosphorylation sites are not. (C) Luciferase assay for TGFβ in MDA-MB231 cells for Par1b loss and rescue with wild-type Par1b or kinase-dead K49A Par1b. Kinase activity of Par1b is not required for its pro-TGFβ effects. Data are represented as mean and SD (D) Smad4 monoubiquitination assay as in Fig. 5A, showing the effect of co-transfected XFrizzled 7 and noncanonical Wnt ligands on Smad4 ubiquitination. (E) The inhibition of Smad4 monoubiquitination caused by Par1b overexpression is not affected by loss of FAM/USP9x. (F) Smad4 ubiquitination assays as is Figure 5A in cells also overexpressing Par1b. Par1b antagonize Smad4 monoubiquitination even in the presence of proteosome inhibitor MG132 (used at 10 μM for 10 hours) suggesting that Par1b does not enhance degradation of ubiquitinated Smad4. Effectiveness of MG132 treatment was monitored by p53 stabilization (data not shown). (G) In-vitro binding of Smad4 to Ecto is inhibited by Par1b alone or in combination with Dvl3. Flag-Smad4 protein (affinity purified) was incubated with Ecto in presence of either recombinant Par1b or HA-Dvl3 (affinity purified) or both. Panels are western blot of Ecto and Dvl3 bound to Smad4 after co-IP with Smad4 antibody. 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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