Figure 2

Domain mapping studies of the interaction between Smad3 and APC10 reveal distinct domains involved in binding. (A) The Smad3C (MH2 domain) is necessary and sufficient for binding to APC10. Top panel: a cartoon to illustrate the deletion constructs of Smad3. The APC10 binding activities of each truncated Smad3 as detected by assays in the bottom panels are summarized at the right side of the panel. Bottom left panel: MH2 domain is necessary and sufficient for Smad3 to bind APC10 in GST pull down assay. Flag-tagged full-length or truncated Smad3, as indicated, was transfected into 293 cells and tested against GST-APC10 and GST (negative control). Bottom right panel: MH2 domain is necessary and sufficient for Smad3 to bind APC10 in 293 cells. Different Smad3 truncations tagged with Flag were co-transfected with T7-APC10 into 293 cells. The expression of these proteins was detected by Western Blot using anti-Flag and the interaction between APC10 and Smad3 truncations was detected by immunoprecipitation of T7-APC10 followed by Western blot using anti-Flag. "L.C." represents the antibody light chain. (B) The C-terminal domain of APC10 is necessary for binding to Smad3. Top panel: a cartoon that illustrates the deletion mutants of APC10. The top three deletion constructs are amino-terminal deletion mutants. The number of amino acids deleted in each construct is indicated. For example, the D2 construct lacks the N-terminal 41 amino acids, thus it is also labeled as D41N. Bottom left panel: the six deletion constructs of APC10, each tagged with Flag, were transfected into 293 cells. The expression of these deletion mutants was detected by Western blot using anti-Flag. Stable protein expression was detected only in cells transfected with two C-terminal deletion constructs (D8 and D9). Bottom right panel: the C-terminal 66 amino acids of APC10 are required for Smad3 binding. T7-tagged APC10 and Flag-tagged APC10 D9 were transfected into 293 cells. Cell lysates were subjected to immunoblotting with anti-T7 (lane 1) and anti-Flag (lane 2) antibody. APC10 and APC10 D9 were tested against GST-Smad3 (lanes 3 & 4) and GST alone as a control (lanes 5 & 6).