FIG 5.

Phosphatase activity toward S384 is decreased in mitosis. (A) Hct116 Fbw7^−/− cells were grown as asynchronous cultures or arrested in prometaphase by adding nocodazole for 18 h. The CDK1/2 inhibitor roscovitine was then added for up to 15 min before harvesting the cells. Endogenous cyclin E was immunoprecipitated, and changes in pS384 were analyzed by Western blotting. (B) HeLa-shFBXW7 cells were treated and processed as described for panel A. (C) Hct116 Fbw7^−/− cells were either grown asynchronously or harvested by mitotic shake-off, following a double-thymidine block and release. Prior to harvesting, cells were treated for 10 min with roscovitine. Changes in endogenous cyclin E S384 phosphorylation were measured by immunoprecipitation followed by Western blotting. (D) Semiquantitative RT-PCR analysis of PPP2R2B (B55β), PPP2R2D (B55δ), and ACTB mRNA expression in Hct116 and HeLa cells.