Fig. 4.

Deleting the MTDH binding site of forkhead box M1(FOXM1) destabilizes the FOXM1 protein. (A) Upper panel, diagram of the domains and mutations of FOXM1. ★, D box; ☆, KEN box; NT, N terminus; DBD, DNA-binding domain; CT, C terminus. Amino acids 180–202 constitute the delta MTDH binding site (DMBS). Lower panel, His pull-down assays were performed using purified GST-tagged MTDH and the indicated truncated or mutant forms of FOXM1. (B) The E1 ubiquitin-activating enzyme UBE1, the E2-conjugating enzyme UBE2C, the E3 ubiquitin ligase APC/C, ubiquitin, and purified GST-tagged Cdh1 were incubated with in vitro-translated MTDH and a truncated or mutant form of FOXM1. The reaction mixture was subjected to IP using an anti-Flag antibody. The reaction product was analyzed by Western blotting for ubiquitin. (C) WT or mutant FOXM1 was cloned into the pBIFC-VN173/Flag vector, and WT MTDH was cloned into the pBIFC-VC155HA vector. The indicated plasmids were co-transfected with the cyan fluorescent protein (CFP) vector into 293T cells. Scale bar, 40 μm. (D) MTDH and WT or mutant FOXM1 were stably expressed in HNA. Total cell extracts were subjected to IP.