Cyclin E sensitivity to Fbw7 varies during the cell cycle. (A) The indicated Hct116 cells were treated with either aphidicolin (S), nocodazole (G2M), or DMSO (A) for 16 h, and lysates were analyzed by Western blot (γ-tubulin, loading control). Cyclin E abundance is shown. In each case, >90% of the nocodazole-arrested cells were in G2M and >70% of the aphidicolin-arrested cells were in S phase (not depicted). (B) The indicated Hct116 cell clones were arrested in S phase with a sequential thymidine/hydroxyurea block and released for the indicated time periods. The abundance of cyclin E, phosphorylated cyclin E species, and Grb2 (loading control) are shown. (C) Hct116 cells were treated as in A and lysates were examined for Fbw7 and cyclin E expression. Fbw7-null cell lysates serve as negative control. (D) Hct116 cells were arrested and released from aphidicolin and Fbw7 abundance was determined.