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Figure 8. ZC4H2 antagonizes Smurfs activity towards Smads by abolishing the association between Smurfs and Smads. (a–d) Effects of ZC4H2 on the polyubiquitination status of Smads mediated by Smurfs. HEK293 cells were transiently transfected with the indicated plasmids and treated for 8 h with MG132 before harvesting. Smad proteins were coimmunoprecipitated with anti-FLAG antibody and then detected for polyubiquitin chains with the antibody against ubiquitin. (e–h) Effects of ZC4H2 on the association between Smurfs and Smad1, Smad1-ΔLinker (e,f) or Smad5 (g,h) proteins. HEK293 cells were transiently transfected with different combinations of expression vectors of ZC4H2 and Smad1, Smad5 or Smad-ΔLinker, as indicated, and treated for 8 h with MG132 before harvesting. Cell lysates were incubated with anti-FLAG beads, washed, and subsequently analysed by western blotting. WCL, whole-cell lysate; IP, immunoprecipitation; IB, immunoblotting; WT, wild-type; ΔL, Smad1-ΔLinker truncates; CA, the ligase dead mutant for Smurf1; CG, the ligase dead mutant for Smurf2. Image published in: Ma P et al. (2017) © 2017 The Authors. 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 license Larger Image Printer Friendly View

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