Figure 6: Neddylation of Smurf1 enhances E2 binding and promotes the substrate ubiquitylation. | Nature Communications

Figure 6: Neddylation of Smurf1 enhances E2 binding and promotes the substrate ubiquitylation.

From: The covalent modifier Nedd8 is critical for the activation of Smurf1 ubiquitin ligase in tumorigenesis

Figure 6

(a) Smad4, Smad7, Smurf1 and Nedd8 were co-transfected into HCT116 cells. Cell lysates were immunoblotted to detect Smad4 levels. (b,c) In vivo ubiquitylation assay of Smad4. The indicated Nedd8 mutants (b) or Smurf1 mutants (c) were co-transfected with Smad4, Smurf1 and ubiquitin into HCT116 cells. Twenty-four hours post-transfection, cells were treated with MG132 (20 μM, 8 h). (d,e) Neddylation enhances ubiquitin E2 binding to Smurf1. Bacterially expressed Ub E2 (UbcH5c or UbcH7) were incubated with in vitro neddylated Smurf1 or unneddylated Smurf1 proteins. Both input and pull-down samples were subjected to immunoblotting with the indicated antibodies. (f) Nedd8 WT or ΔGG mutant was incubated with UbcH5c, Smurf1 and the neddylation E1/E2. Interaction between Smurf1 and UbcH5c was measured.

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