Figure 2. Cdc28 inhibition causes a... | Rockefeller University Press

Figure 2.

$Figure 2. Cdc28 inhibition causes a decrease of Cdc20 levels during a checkpoint arrest. (A–D) CDC28 PDS1-MYC18 (yAC359) and cdc28-as1 PDS1-MYC18 (yAC779) cells were synchronized in G1 phase with α-factor and then released in YEPD supplemented with nocodazole. After 3 h in nocodazole, the indicated concentrations of 1-NMPP1 or DMSO were added together with 20 µg/ml of α-factor. Samples were taken at the indicated times after the addition of 1-NMPP1 or DMSO for Western blotting. (E, top) cdc28-as1 MAD2-3GFP NUF2-mCherry (yAC2182) cells were treated similarly to A–D: after 2.5 h in nocodazole (noco), cells were kept two additional hours in the presence of nocodazole supplemented with DMSO, 500 nM, or 5 µM 1-NMPP1 (120 min). One fraction of cells was instead released from nocodazole (Release). Colocalization of Mad2 with Nuf2 was scored at 120 min (see also Materials and methods); error bars refer to standard errors from three independent experiments. In each experiment, 50 cells were analyzed for each condition. (E, bottom) Examples of localized and nonlocalized Mad2.$

Cdc28 inhibition causes a decrease of Cdc20 levels during a checkpoint arrest. (A–D) CDC28 PDS1-MYC18 (yAC359) and cdc28-as1 PDS1-MYC18 (yAC779) cells were synchronized in G1 phase with α-factor and then released in YEPD supplemented with nocodazole. After 3 h in nocodazole, the indicated concentrations of 1-NMPP1 or DMSO were added together with 20 µg/ml of α-factor. Samples were taken at the indicated times after the addition of 1-NMPP1 or DMSO for Western blotting. (E, top) cdc28-as1 MAD2-3GFP NUF2-mCherry (yAC2182) cells were treated similarly to A–D: after 2.5 h in nocodazole (noco), cells were kept two additional hours in the presence of nocodazole supplemented with DMSO, 500 nM, or 5 µM 1-NMPP1 (120 min). One fraction of cells was instead released from nocodazole (Release). Colocalization of Mad2 with Nuf2 was scored at 120 min (see also Materials and methods); error bars refer to standard errors from three independent experiments. In each experiment, 50 cells were analyzed for each condition. (E, bottom) Examples of localized and nonlocalized Mad2.