TI - Smad3 interaction with APC10 is regulated by TGF-beta type I receptor activation while CDH1 interaction with HEF1 is constitutive . AB - To further test the effect of TGF-beta receptor activation on the interaction between Smad3 and APC10 or between CDH1 and HEF1 , these proteins were expressed in 293 cells in the presence or absence of a constitutively activated TGF-beta type I receptor R4T204D ( R4TD ) . As shown in Figure 5A , APC10 was detected only in the immunoprecipitates of Smad3 from cells with the coexpression of both Smad3 and R4TD ( Fig 5A , lane 6 ) . We noted that anti-Smad3 precipitates two forms of Smad3 in lane 6 while only one form of Smad3 in lane 5 . The higher molecular weight form is a phosphorylaTED version of Smad3 , as confirmed by Western blot with anti-PHOSphoserine ( data not shown ) . Thus , the data suggests that TGF-beta receptor -induced Smad3 PHOSphorylation may enhance its interaction with APC10 . This appears to be not the case for HEF1 interaction with CDH1 . As shown in Figure 5B , the complex formation of CDH1 and HEF1 occurs in the absence of R4TD and the coexpression of R4TD did not alter the formation of the complex ( Fig 5B , compare lanes 9 & 10 in top panel ) . The coexpression of Smad3 with CDH1 and HEF1 caused significant reduction of HEF1 protein levels , as expected , but also did not alter the complex formation of CDH1 and HEF1 ( Fig 5A , lanes 11 & 12 ) . When HEF1 was coexpressed with CDH1 , APC10 and the MH2 domain of Smad3 , which harbors the binding sites for both HEF1 and CDH1 , we detected al four proteins in the immunoprecipitates of HEF1 ( Supplementary Fig 1 ) . These data further confirms the complex formation of Smad3 , HEF1 , CDH1 and APC10 in mammalian cells and also suggest that TGF-beta signaling may regulate the complex formation via regulating the interaction between Smad3 and APC10 .