Fig. 3
From: MAPK-dependent control of mitotic progression in S. pombe
Pmk1 physically interacts with Slp1CDC20 through a MAPK docking site. A Mitotically arrested nda3-KM311 cells (6 h) were harvested and Pmk1- Cdc20Slp1 physical interaction determined by Co-IP. Untagged Cdc20Slp1 and Pmk1 were used as negative controls. IP: Immunoprecipitation; IB: Immunoblotting. Representative image of three independent experiments. B Slp1-HA was immunoprecipitated from the mitotically arrested nda3-km2311 (pek1+) and nda3-km311 pek1Δ cells (6h) and its physical interaction with Pmk1 determined by western blotting. Representative image of two independent experiments. C Schematic representation of Slp1 protein indicating a putative MAPK docking site at its N-terminus. Slp1 MAPK Docking site was mutated creating the Cdc20Slp1-DSmutant. Pmk1 physical interaction with Cdc20Slp1and Cdc20Slp1-DSwas tested by Co-IP experiment from mitotically arrested cells. D Cdc20Slp1 levels from asynchronously growing cells from the indicated strains. Numbers indicate fold change relative to wild type using Cdc2 as loading control. E Representative example of mitotic arrest assay of the indicated strains. Percentage of septation is shown as readout for mitotic exit/leakage; n=200 cells were quantitated for each strain and time point. C–E Representative examples of three independent experiments. F Cdc20Slp1-DS mutant was tested by Co-IP experiment for its ability to interact with the MCC components Mad2 (above) and Mad3 (below) during a mitotic block. Untagged Cdc20Slp1-DS, Mad2, and Mad3 were used as negative controls. Representative example of two independent experiments