FIG. 6.

Wild-type and phospho-mutant AML1 display different stabilities after cycloheximide treatment. (A) Endothelial cell lines expressing wild-type AML1, AML1-4A, or AML1-4D were treated for 0, 8, or 16 h with 20 μg/ml cycloheximide (CHX) as indicated above the lanes. Before lysis, the number of cells in each sample was determined, after which whole-cell lysates were prepared from each sample. Lysate from an equal number of cells was loaded into each lane, and the level of AML1 in each sample was determined by Western blotting with anti-AML1 antibodies. Ponceau staining of the membrane after transfer from the SDS gel was used to determine the amount of total protein in each sample. The relative amount of wild-type AML1, AML1-4A, or AML1-4D in each sample was calculated by densitometry, and the value was normalized to the amount of total protein (values are given beneath the upper panel). (B) Endothelial cells expressing HA-tagged wild-type AML1 were treated for 0, 8, or 20 h with 20 mg/ml cycloheximide (CHX) as indicated above the lanes. AML1 was then immunoprecipitated with anti-HA antibodies and analyzed by Western blotting with anti-serine 303-phosphorylated AML1 antibodies and anti-AML1 antibodies.