Fig. 6. REF-52 cells induced to permanently arrest by aphidicolin contain markers and activity consistent with S phase arrest. (A) Protein expression levels. REF-52 cells released from G0 arrest were treated with aphidicolin (10 µM) for the indicated times, then harvested. To determine expression levels of various cell cycle proteins, samples were subjected to SDS-PAGE, and then exposed to the appropriate antibodies for western blots. (B) Cyclin-dependent kinase activity. The kinase activities of Cdk2 and Cdc2 were determined in similar cell extracts, following specific immuneprecipitation of the enzymes, using histone H1 as substrate. Autoradiographs of ³²P incorporation from [-³²P]ATP are shown. (C) Release of MCM3 and MCM4 from the nuclear matrix fraction during prolonged arrest in S phase. MCM proteins were analyzed for their localization to the nuclear matrix when treated with aphidicolin. For analysis of prolonged S phase block, G0 synchronized REF-52 cells were treated with aphidicolin for the times indicated. Immunoblots of MCM3 and MCM4 proteins in chromatin-bound and soluble fractions are shown. The soluble fractions contain both cytoplasmic and soluble nuclear proteins. Cdc25A, which remains constant in the two fractions, was used as a loading control. (D) The progressive release of MCM3 from the nuclear matrix fraction to the soluble fraction during normal S phase progression is shown as a control. For this analysis, REF-52 cells were synchronized at the G1/S boundary by 30 hours treatment with aphidicolin, released into drug-free medium, and then extracts were prepared every 2 hours during S phase recovery.