FIG. 9.

AML1 associates with Cdh1, Cdc20, and Skp2, and the AML1 destruction boxes are required for efficient association with Cdc20 but are not required for Cdh1. (A) 293T cells were transfected with the indicated expression plasmids and lysed in PBS-1 mM EDTA-0.5% Triton X-100. Excess AML1 expression vector was used for cotransfections to ensure overexpression of AML1 relative to Cdh1, Cdc20, or Skp2. This was done to make sure that induced degradation of AML1 was not observed and confused with any effects phosphorylation might have on the association of AML1 with Cdh1, Cdc20, or Skp2. A total of 200 μg of each sample was immunoprecipitated (IP) with the indicated antibodies, and immunoblotting was performed with anti-AML1 followed by anti-myc or anti-HA. Ten micrograms of each lysate was also resolved by SDS-PAGE and immunoblotting was performed as described above to determine the level of protein expression in every sample. (B) 293T cells were transfected with the indicated plasmids and the experiment performed as described above. DBM, destruction box mutant.