Distinct and sequential re-replication barriers ensure precise genome duplication
Fig 3
Cdt1 hyperphosphorylation is dependent on linker sites and CDK1 activity.
A) Workflow for U2OS cell line synchronization and inhibitor treatment; inhibitors are indicated in 3C. B) Whole cell lysates were separated by Phos-tag SDS-PAGE (top) or standard SDS-PAGE (middle) followed by immunoblotting for ectopic Cdt1 (HA); total protein stain serves as a loading control. C) Cells were synchronized with nocodazole as in A, then mock treated or treated with 10 μM RO-3306 (lane 2), 6 μM CVT313 (lane 3), 30 μM SB203580 (lane 5), 10 μM JNK inhibitor VIII (lane 6), or combinations of inhibitors as indicated for 1 hour except that RO3306 treatment was for only the final 15 minutes to preserve mitotic cell morphology. All cells were simultaneously treated with 20 μM MG132 to prevent premature mitotic exit. Endogenous Cdt1 phosphorylation was assessed by standard or Phos-tag SDS-PAGE followed by immunoblotting; total protein stain serves as a loading control. The example shown is representative of more than three independent experiments.