Cells were grown to mid-log phase at the permissive temperature (23°C) and arrested in G1 with 10 μg/ml alpha-factor for three hours (with a second equivalent amount added after two hours). Cells were then released into medium without alpha-factor at the restrictive temperature (30°C) to inactivate ycg1-2. Cells were collected at 60-, 75-, and 90-minutes following release to enrich for anaphase cells. Samples were fixed and stained as previously described [61 (link)], with minor modifications. Permeabilized cells were stained with anti-tubulin (YOL1/34, Invitrogen) and anti-Nop1p primary antibodies (Encor) for two hours, and anti-rat FITC (Jackson Immunoresearch) and goat anti-mouse Alexafluor 594 (Invitrogen) secondary antibodies for two hours, both at room temperature. Cells were imaged on a Nikon Eclipse E400 microscope. To ensure that cells at similar stages of anaphase were being compared, late anaphase cells were identified as those with an elongated spindle and clearly segregated DAPI masses. Nop1 segregation was then scored as equal or unequal (either absent or uneven) in only late anaphase cells. A minimum of 100 cells per strain were scored in each experiment.
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