Figure 1

Smad3 interacts with APC10 specifically. (A) Co-immunoprecipitation of Smad3 and APC10 from mammalian overexpression system. 293 cells were transiently transfected with T7-APC10 and Smad proteins as indicated. The cell lysates were subjected to Western blot analyses directly or immunoprecipitation followed by Western blot as indicated. (B) Smad3 binds to APC10 in GST pull down assays. Flag-tagged Smad constructs were transfected into 293 cells and tested against GST-APC10 purified from E. Coli. Total input Smad protein levels (100 ug) were detected by immunoblotting of the cell lysates with anti-Flag antibody (top panel). Smad proteins bound to the GST-APC10 beads were eluted and analyzed by immunoblotting with anti-Flag antibody (middle panel). As a control, F-Smad proteins were incubated with the GST alone (bottom panel). (*) We noted that GST-APC10 was recognized by the anti-Flag antibody on Western blot. (C) Smad3 binds to HEF1 and APC10 in in vitro binding assay. In vitro translated ³⁵S-labeled HEF1, ³⁵S Smad3 and ³⁵S APC10 were incubated with GST-Smad3, GST-APC10 or GST as indicated. The in vitro translated proteins before and after binding were detected by electrophoresis and autoradiography.