Figure 4

CDH1 interacts with HEF1 at the HEF1 C-terminal M2 domain. (A) In vitro binding test. In vitro translated ³⁵S labeled CDH1 was incubated with GST-HEF1 or GST alone as a control in modified lysis buffer. ³⁵S labeled CDH1 was separated by electrophoresis and detected by autoradiography. (B) A cartoon that illustrates putative D boxes on HEF1. (C) A cartoon that illustrates the deletion constructs of HEF1 used in (D). (D) Co-immunoprecipitation of HEF1/HEF1 deletions with CDH1 in 293 cells. Full-length HEF1 and T7-tagged HEF1 deletions were co-transfected with myc-tagged CDH1 into 293 cells. Myc-CDH1 was immunoprecipitated with anti-myc antiboby and CDH1 bound HEF1 or HEF1 deletions were detected by immunoblot with either anti-p130Cas antibody (top panel, lanes 1 & 2) or anti-T7 antibody (top panel, lanes 3–5). The expression level of CDH1 was detected with anti-myc antibody (second panel). The amount of HEF1 (third panel) or T7-HEF1 deletions (bottom panel) expressed was detected by anti-p130Cas for HEF1 or anti-T7 antibody. (E) A cartoon that illustrate the putative complex of HEF1, Smad3, APC and CDH1.