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. 2016 Dec 30;205(3):1169–1178. doi: 10.1534/genetics.116.195727

Figure 3.

Figure 3

The abolishment of Bub1-dependent H2A phosphorylation leads to premature SAC silencing. (A) h2a-S121A mutant cells are sensitive to CIK1-CC overexpression. WT and h2a-S121A cells with a vector (V) or a PGALCIK1-CC (CC) plasmid were serial 10-fold diluted and then plated onto glucose and galactose plates for further incubation at 30° for 2 days. (B) h2a-S121A mutants lose viability after CIK1-CC overexpression. Cells with the indicated genotypes in raffinose medium were released into galactose medium at 30°. Cells were collected at 0, 2, 4, and 6 hr and spread onto YPD plates to examine the plating efficiency after overnight growth at 25° (n ≥ 200). (C) h2a-S121A mutation abolishes the anaphase entry delay in dam1-3D mutants. Cells with the indicated genotypes were arrested in G1 phase with α-factor and then released into cell cycle in YPD. Cells were collected over time to examine the Pds1 protein levels. Budding index and Pds1 protein levels are shown. Pgk1, loading control. (D) h2a-S121A mutant cells show efficient metaphase arrest in response to nocodazole treatment. G1-arrested PDS1-18myc and h2a-S121A PDS1-18myc cells were released into YPD medium containing 20 μg/ml of nocodazole and incubated at 30°. Pds1 protein levels were determined after Western blotting. The Pds1 levels and budding index are shown. Pgk1, loading control.