Regulation of Ndd1 turnover.

<p>A) Cells expressing Ndd1-Flag from its endogenous promoter were arrested in G1 (with 10 μg/ml α–g/mlh (αF)), metaphase (with 10 μg/ml nocodazole (noc)), or exposed to DNA damage (with 0.05% MMS) for 2.5 hours. Cycloheximide (CHX) was added at 50 μg/ml at t = 0 and protein turnover was followed with 15 minute timepoints. Rad53 is shown as a control for damage treatment, because it hyper-shifts in response to DNA damage. B) Expression of Ndd1-Flag from the <i>GAL1</i> promoter (<i>GAL1</i>p-Ndd1-Flag) was induced by growth in galactose for 6.5 hours until cells reached mid-log phase. Cells were treated with αF, noc, or MMS for 2.5 hours and turnover was followed as in [A]. Cdc28 is shown as a loading control. C) Quantification is shown of samples in [B]. On the left, quantification is shown from lighter exposure. Right panel shows quantification of αF samples only from the darker exposure. Y-axis shows the normalized signal above background normalized to the value at t = 0 in nocodazole arrested cells (left) or αF arrested cells (right). D) Cells expressing <i>GAL1</i>p-Ndd1-Flag were induced by growth in galactose for 3 hours, then arrested in G1 with 10 μg/ml αF for 2.5 hours, and released into media containing either 10 μg/ml nocodazole or 0.2 M hydroxyurea (HU). Cycloheximide was added after 1.5 hours and protein turnover was followed with 15 minute timepoints. FACS analysis shows G1 peak from the αF arrest and cell cycle progression following 1.5 hours release into HU and noc. E) <i>GAL1</i>p-Ndd1-Flag was induced with galactose until cells reached mid-log phase. Cells were arrested in G1 with αF for 2.5 hours, and released into the cell cycle and Ndd1 was examined. αF was re-added to the media after the 30 minute timepoint was collected to prevent the cells from entering a second cell cycle. Clb2 levels are shown to indicate cell cycle position. Cdc28 is shown as a loading control. F) FACS analysis showing cell cycle progression of timepoints shown in [F].