Mps1Mph1 Kinase Phosphorylates Mad3 to Inhibit Cdc20Slp1-APC/C and Maintain Spindle Checkpoint Arrests (original) (raw)
Fig 3
MCC assembly defects are rather minor in the mad3-C9A and mad2 mad3 double phospho-mutants when compared to mps1-kd.
A. The cdc25 strains indicated were pre-synchronised in G2 by shifting to 36°C for 3.5 hours. They were then released at 25°C and time points taken every 15 minutes. Native extracts were made and Cdc20Slp1 was immunoprecipitated with anti-Flag antibodies. Immunopreciptates were separated by SDS-PAGE and immunoblotted for Cdc20Slp1, Mad3p (anti-GFP) and Mad2p. B. These immunoblots were quantitated and plotted as the intensity of Mad3 or Mad2 relative to the Cdc20Slp1 level at each time point (45 and 60 minutes after release into mitosis). All values were then compared to wild-type–see Materials and Methods for details. C. Cell cycle progression was scored by DAPI staining methanol fixed cells and quantifying the % of bi-nucleate cells in the population at each time point (200 cells were scored at each time point).