Fig. 5
From: MAPK-dependent control of mitotic progression in S. pombe
Cdc20Slp1 downregulation induced upon osmotic stress is prevented in the absence of Mad3 or proteasome activity. A Asynchronously growing cells treated with 0.6M KCl for the indicated times were harvested and Slp1-HA was immunoprecipitated and tested for its ability to bind Mad3. Time 0’ represents the Cdc20Slp1-Mad3 association before KCl addition. IP: Immunoprecipitation; IB: Immunoblotting. Quantification of Cdc20Slp1 and Mad3 levels from two independent experiments shown in left. B Left,Cdc20Slp1 protein levels were determined from asynchronously growing wild type and mad3Δ cells treated or untreated with 0.6M KCl at 25°C. Cdc2 was used as a loading control. Right, Average Cdc20Slp1 protein levels relative to time 0 from three independent experiments. Asterisk denotes p<0.001. ns: non-significant. C nda3-KM311 cells were arrested in mitosis for 7 h and KCl, or KCl plus proteasome inhibitors (MG132 plus Bortezomib) were added to the cultures. Levels of Cdc20Slp1 were analyzed at indicated time points by western blot. Cdc2 was used as loading control. Image shows a representative example of two independent experiments. Fold change in Cdc20Slp1 levels relative to control from the images on the left is indicated. D mts3-1 cells grown at permissive and restrictive temperatures were treated with KCl 0.6M. After 30 min of KCl addition Slp1 was immunoprecipitated in each condition and the presence of poly-ubiquitinated forms of Slp1 was detected using an anti-ubiquitin antibody. ECdc20Slp1 or Cdc20Slp1-DS proteins were electrophoretically separated using 10µM of Phostag. F Schematic representation of MAPK-dependent Slp1 downregulation both in unperturbed cell cycles and during environmental stress. D–E Representative example of two independent experiments