Cyclin E–CDK2 activity varies during the cell cycle. (A) HeLa cells were transfected with either control or Fbw7-siRNA and then 48 h later were treated with nocodazole for 16 h. Mitotic cells were shaken off and replated for the indicated times. Cyclin E abundance and activity, as well as cyclin A abundance (a marker of cell cycle synchronization) are shown (*, background band). (B) Hct116 cells or Fbw7-null Hct116 cells were transfected with either control or p21Cip1-specific siRNAs and, after 48 h, treated with aphidicolin (Aph) or nocodazole (Noc). Cyclin E abundance, activity, and S384 phosphorylation are shown. The efficacy of p21 knockdown is shown. (C) Fbw7-null Hct116 cells were treated with aphidicolin or nocodazole and lysates were immunoprecipitated with anti–cyclin E antibody. The amount of CDK2 and Y15–phosphorylated CDK2 bound to cyclin E is shown. (D) Increased Fbw7 binding of cyclin E obtained from mitotic Fbw7-null lysates. Compare the amount of cyclin E that coprecipitates with Fbw7 in lanes 4 and 5 (middle).