Figure 6

Both APC10 and CDH1 can alter the steady state protein levels of HEF1 (A) Overexpressed APC10 caused the reduction of the steady-state protein levels of HEF1 and such an activity is compromised in a deletion mutant of APC10 (APC10 D8) that is defective in binding to Smad3. HEF1 was co-transfected with ubiquitin, Flag-Smad3, T7-APC10 or T7-APC10 deletion mutant APC10 D8. The expression level of HEF1 was detected by Western blot using anti-p130Cas antibody, as shown in the top panel; the expression of Smad3, APC10 and APC10D8 were detected by Western blot using anti-Flag and anti-T7, as shown in the bottom panel. (B) CDH1 enhances Smad3-regulated HEF1 degradation. HEF1 was co-expressed in different combinations with myc-CDH1, T7-APC10, Flag-Smad3 and R4T204D (R4TD), a constitutively active type I receptor in 293 cells. The steady state levels of HEF1 were detected by anti-HEF1 antibody (top panel) and the expression levels of Smad3 and APC10 were detected with anti-Flag and anti-T7 antibodies, respectively (second and third panels), while the expression levels of myc-CDH1 were detected by anti-Myc (fourth panel). To detect R4-TD, cell lysates were blotted with anti-TGF-β RI antibody (bottom panel).