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. 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). Plotted as mean with standard deviation (n = 2 experiments). C. Methanol-fixed cells were stained with calcofluor and scored for septation, indicating a failure to maintain spindle checkpoint arrest. This experiment was repeated twice and >100 cells scored for each strain at each time point. Plotted as mean with SEM.