Figure 5

The formation of a ternary complex between Smad1, Az and HsN3. A. Az interacts with Smad1 and the interaction is enhanced by receptor activation in the presence of lactacystin. COS cells were transfected with the indicated plasmids. F-Smad1 was precipitated with anti-Smad1 monoclonal antibody and the co-precipitated F-Az was detected by anti-Flag Western blot. "IgG.L." represents the IgG light chain. The total expression of the proteins in the lysates was detected by Western blot as shown in the bottom panels. B. Results of domain mapping analyses of Az. Different deletion constructs of Az were made as illustrated and subcloned into yeast expression vector pJG4-5, which were then co-transformed with full length LexA-Smad1 and LexA-HsN3 into yeast two-hybrid system. The interaction tests were carried out similarly as shown in Fig. 1. The separate domains important for Az to bind HsN3 and Smad1 are indicated. C. Az interacts with both Smad1 and HsN3 when they are co-expressed at a specific ratio. COS1 cells were co-transfected with the indicated constructs of Smad1, Az and HsN3. The amount of DNA used for the transfection is also indicated. The co-precipitated Smad1 and Pro-N3-F are marked. D. A cartoon to summarize the observed complex formation between Smad1, HsN3 and Az along the proteasome assembly pathway.