Figure 6From: Direct interaction between Smad3, APC10, CDH1 and HEF1 in proteasomal degradation of HEF1Both 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).Back to article page