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. 2017 Feb 7;7:41364. doi: 10.1038/srep41364

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Copyright © 2017, The Author(s)

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(A,B) The mutation in the Smurf Nedd8 binding interferes with RhoA or Smad1 targeting in cells. HEK293T cells were transiently transfected with Flag-tagged RhoA or Myc-tagged Smad1 in combination with Myc-tagged Smurf1 WT, Y439A or 10 A mutant. Steady-state levels of RhoA, Smad1 and Smurf1 in total cell lysates were determined by immunoblotting with anti-Flag and Myc antibodies. (C,D) Mutation in Smurf1 binding interferes with the ubiquitylation of RhoA or Smad1 in regards to targeting in cells. HEK293T cells were transiently transfected with Flag-tagged RhoA or Smad1 together with Myc-tagged Smurf1 WT or 10 A as indicated. Ubiquitylation of RhoA or Smad1 was determined by immunoprecipitation by anti-Flag followed by immunoblotting with anti-ubiquitin antibody. (E) Analysis of the interaction between Flag-tagged RhoA and Myc-Smurf-HECT (WT or the 10 A mutant) by Co-IP in vivo.