Bub3 is depleted from the nucleus upon rapamycin addition. (A) Representative time lapse of Bub3-FRB-GFP cell wherein rapamycin is added at t = 0 min to visualize when Bub3 is anchored away from the nucleus (when GFP focus was no longer visible). Bub3-FRB is tagged with GFP and Spc42 is tagged with mCherry. Scale bars, 5 µm. (B) Graph of the mean time of Bub3 nuclear depletion after rapamycin addition. 100 cells from two independent experiments were monitored. Error bars represent SEM. (C) Mitotic growth of different strains with and without rapamycin and benomyl addition. Cultures are serially diluted 1:10 from a saturated yeast culture. Rapamycin does not affect the mitotic growth of the anchor-away strains due to their genetic background (tor1-1 mutation). However, the strains without the tor1-1 mutation are sensitive to rapamycin (wild-type, bub3Δ, bub1Δ, and sgo1Δ). Final concentrations of benomyl and rapamycin were 15 µg/ml and 1 µg/ml, respectively. The plates were incubated at 30°C for 36–40 h before imaging. aa, anchor away.