Figure 4

Loss of Csn5 results in an increase in cyclin E, an F-box protein substrate. (A) Substrates of the Fbw7 SCF complex accumulate when Csn5 is depleted. Cells were induced with doxycycline for eight days followed by SDS-PAGE/western blot analysis for the indicated proteins. (B) Activity of the protein kinase cyclin E/Cdk2 is increased when cells are depleted for Csn5. Cells were induced with doxycycline for eight days followed by lysis under native conditions. Lysates were immunoprecipitated with antibodies toward cyclin E and immuoprecipitates analyzed for kinase activity against histone H1. (C) Cell cycle regulation of cyclin E is lost when cells are depleted for Csn5. Cells were induced with doxycycline for eight days followed by arrest at the indicated cell cycle stages. Cell lysates were analyzed by SDS-PAGE/western blot with the indicated antibodies.