FIG.4.
Smad4 MH1 domain mediates its interaction with multiple regions of Skp2. (A) Mapping of Smad4-interacting domain on Skp2. 293T cells were transfected with Flag-tagged Smad4(R100T) and HA-tagged Skp2 deletion constructs. Skp2-bound Smad4 was detected with anti-HA immunoprecipitation coupled with anti-Flag immunoblotting. The levels of transfected proteins in whole-cell lysates (WCL) are shown (bottom panels). (B) Mapping of Skp2-interacting domain on Smad4. 35S-labeled Smad4 deletion mutants were generated with the TNT kit (Promega) and then incubated with immobilized GST-Skp2 protein. Smad4 fragments bound to Skp2 (left) and their inputs (right) are shown. (C) Schematic diagrams of Skp2 and Smad4 constructs. Deletion mutants of Skp2 and Smad4 are shown with color-coded domains. For Skp2, F indicates the F box and LRR indicates leucine-rich repeats. For Smad4, the MH1, linker, and MH2 domains are shown. The Skp2-Smad4 interaction is summarized on the right.