Figure 3. AKT1 enhances Twist1 ubiquitination and degradation.

(A) Protein expression of Twist1 in wild-type and AKT1-deficient MEFs.

(B) Overexpression of myr-AKT1 and myr-AKT2 in HeLa cells by retrovirus infection. Cell lysates from these stable clones were subjected to Western blotting with the indicated antibodies.

(C) Phase contrast imaging of EMT morphotypic changes in MCF 10A shCTRL, shAKT1, and shAKT2 stable cells treated with 100 nM TGFβ for 6 h. Protein expression was analyzed by Western blot (right).

(D) Western blot analysis of Twist1 protein stability with indicated transient transfection. Cells were then treated with 20 mM cycloheximide as the indicated intervals.

(E) Protein half-life of Twist1 WT, AVA and DDD was determined as described in Fig. 3D.