Figure 3

Smad3 MH2 domain contains overlapping but distinct binding sites for HEF1 and APC10. (A) A Cartoon to illustrate the deletion constructs of Smad3 (Smad3d2, d4 and d6) as well as a hybrid protein S3/S1/S3. (B) GST pull-down assay to map APC10 binding site on Smad3C. Flag-tagged Smad3 deletion mutants were transfected into 293 cells and the cell lysates were tested against GST-APC10. Cell lysates (top panel) and proteins bound to the beads (middle and bottom panels) were analyzed by Western blot with anti-Smad1/2/3 from Santa Cruz. The percentage listed at the right side of the listed constructs is derived from dividing the signals (measured by ImageQuant) of bound proteins in the middle panel by the signals of lysate proteins in the corresponding lane of the top panel and then times 100 percent. (C) GST pull-down assay to map HEF1 binding site on Smad3C. Same as in (B), with GST-APC10 replaced by GST-HEF1. (*) endogenous Smad1 and Smad3 recognized by anti-Smad1/2/3 antibody. (D) A cartoon to illustrate the complex of Smad3, HEF1 and APC10 and their potential interaction with the APC ligase core complex.