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Figure 5. Dvl and Par1b regulate Smad4 monoubiquitination. (a) Western blot analysis detecting Smad4 monoubiquitination in lysates from HEK293T cells transfected with HA-ubiquitin, Flag-Smad4 and the indicated effectors of the Wnt pathway, that is, mFrizzled8 and hDvl3. Ub-Smad4 is a 75-kDa band reactive to the anti-Smad4 antibody (here shown) and to anti-HA (data not shown; see Dupont et al.6). (b) Western blot analysis of Smad4 monoubiquitination as in (a) comparing the activity of Par1b wild-type and kinase-dead K49A mutant (40 ng DNA/cm2). (c) Anti-Smad4 immunoprecipitation and western blot analysis of lysates of MDA-MB-231 cell transfected with HA-ubiquitin, Flag-Smad4 and the indicated siRNAs. Validation of effective knockdown of Par1b and FAM was carried before ubiquitination assays (data not shown). (d) Smad4 monoubiquitination detected as in (a) in lysates from control and Par1b-depleted cells, transfected with two doses of mFz8 expression vector (5 and 10 ng/cm2). (e) co-IPs for Smad4 from HEK293 T cells. Endogenous binding of Smad4 to Ecto is inhibited by overexpression of Dvl3 or Par1b. (f) co-IP of endogenous Smad4 and Ecto proteins is enhanced in HEK293T cells depleted of Par1b. (g) co-IP of Smad4 with Smad2/3, monitoring the formation of the active Smad complex, induced by TGFβ in HEK293T cells transfected with the indicated siRNAs. (h) Dvl and Par1b modulate Ecto activity. Graphs are luciferase reporter assays stimulated by transfection of constitutive-active-TGFβ receptor (CA-ALK5) in MDA-MB-231 cells in presence of the indicated expression constructs. Data are represented as mean and S.D. (*P<0.05 Student's t-test) 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 |