FIG. 1.

Cbk1 is phosphorylated at the HM site upon release from nocodazole arrest. (A) HM site phosphorylation is detected after release from nocodazole arrest. Wild-type (CBK1) cells were arrested for 2 h in nocodazole and released into fresh medium at 30°C. After 70 min, fresh nocodazole was added to arrest cells in the following mitosis. Cells were harvested at the indicated times, and immunoprecipitated Cbk1 was probed with an anti-pT743 antibody (top panel) or anti-Cbk1 antibody (bottom panel). The percentage of Cbk1 phosphorylated at T743 is shown below each lane. (B) HM site phosphorylation occurs at the M/G₁ transition just prior to bud emergence. The budding index upon release from nocodazole arrest is shown over time. (C) Ace2 target gene expression correlates with HM site phosphorylation. RNA was harvested from cells collected at the indicated times, and transcript levels were determined by QPCR. The increases in CTS1 and DSE1 transcripts relative to nocodazole arrest (time zero) are indicated. Standard errors of the means (SEM) for two experiments are shown. (D) Quantification of Cbk1 by immunoblotting. A standard curve was generated by plotting pixel intensity (Odyssey) to picomoles of Cbk1. Error bars represent errors of three independent immunoblot assays. A representative blot is shown. (E) Quantification of pT743 by immunoblotting. A standard curve was generated by plotting pixel intensity (Odyssey) to picomoles of phosphorylated Cbk1. Error bars represent results from three independent immunoblot assays. A representative blot is shown for the phosphorylated peptide and a nonphosphorylated control.