Mps1Mph1 Kinase Phosphorylates Mad3 to Inhibit Cdc20Slp1-APC/C and Maintain Spindle Checkpoint Arrests
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. The anti-microtubule drug carbendazim (CBZ) was added after 20 minutes. Extracts were made, Apc4-TAP pulled down with IgG Dynabeads, separated by SDS-PAGE and immunoblotted for associated Mad3p and Mad2p. B. The immunoblots were quantitated for Mad3p and Mad2p levels, normalised to Apc4p levels, and then plotted relative to the wild-type levels at the 45 and 60 minute time points (after release into mitosis). C. At each time point cells were fixed in 100% methanol and later stained with DAPI and calcofluor to score septation (200–300 cells were scored per sample, per time point). Septation indicates a failure to maintain the spindle checkpoint arrest.