Figure 1. Smurf2 interacts with Nedd8 both in vitro and in vivo.

(A) Direct interaction between Nedd8 and Smurf2 was revealed by GST pull-down assays. Both input and pull-down samples were subjected to immunoblotting with anti-His and anti-GST antibodies. Input represents 20% of that used for pull down. (B) Co-immunoprecipitation (Co-IP) of endogenous Nedd8 and endogenous Smurf2 from HEK293 cells. Western-blot analysis of whole-cell lysates and immunoprecipitates with Smurf2 antibody or control IgG. To avoid Smurf1 degradation, proteasome inhibitor, MG132 was added and incubated for 12 h before cells were harvested. IP, immunoprecipitate; IB, immunoblotting; IgG, immunoglobulin; IgG HC, IgG heavy chain. IgG LC, IgG light chain. (C) Smurf2 was co-immunoprecipitated with both wild type and ΔGG mutant forms of Nedd8 but did not interact with the mutant of I44A. HEK293T cells transfected with Myc-Nedd8 or the mutants and Flag-Smurf2 were immunoprecipitated with anti-Flag, followed by immunoblotting analysis. (D) Mapping of interacting regions of Smurf2 with Nedd8. Flag-Nedd8 and the indicated Smurf2 truncates were coexpressed in HEK293T cells. Cell lysates were incubated with anti-Flag antibody to precipitate Smurf2 deletion mutants. Both the lysates and the immunoprecipitates were analyzed using western blot analysis with the indicated antibodies.