FIG. 7.

APC targeting subunit Cdc20 promotes the degradation of AML1 in a phosphorylation-dependent manner. (A) 293T cells stably expressing wild-type AML1, AML1B-4A mutant, or the phospho-mimic AML1B-4D mutant protein were transfected with either empty vector (control) or vector expressing Cdh1, Cdc20, or Skp2. At 24 to 48 h after transfection, cell lysates were prepared and used for Western blotting with anti-AML1 antibodies, followed by anti-HA antibodies to detect HA-tagged Cdh1 or Cdc20. Samples were stained with Ponceau solution after transfer to membranes to confirm approximately equal loading. (B) Three independent transfection experiments were performed as described for panel A using Cdh1, Cdc20, and Skp2. The quantity of wild-type AML1, AML1-4A, or AML1-4D was determined by densitometry of the bands visualized with the anti-AML1 antibodies. Differences in sample loading were corrected by measuring the intensity of the protein bands stained with Ponceau solution. The resulting values are presented graphically, with the amount of AML1 in the sample transfected with empty vector set at 1.0.