Fig. 4

Stress elicits a rapid drop in Cdc20^Slp1 protein levels, thus delaying mitotic exit. A Left, Exponentially growingwild type cells were exposed to 20µg/ml MBC and Cdc20^Slp1 levels were determined at the indicated time points. MAPK activation was determined using anti-phospho p42/44 and anti-phospho p38 as indicative of respective Pmk1 and Sty1 activation. Right, Cdc20^Slp1, Mad2, and Mad3 levels from asynchronously growing wild type cells were determined in control cells or after 30 min of KCl addition (0.6M). MAPK activation was determined using anti-phosho-p44/42 antibody and anti-phospho p38 as indicative of respective Pmk1 and Sty1 activation. Representative example of two independent experiments is shown. B Left, Cdc20^Slp1 levels in cdc25-22 slp1-HA cells blocked (for 3.5 h) and then released to the permissive temperature (25°C) in unperturbed conditions (left) or after addition of KCl at time 65 min. Right, Relative Cdc20^Slp1 levels and septation index over the course of the experiment. Representative example of three independent experiments is shown. C Left, Cdc20^Slp1 levels from asynchronously growing wild type, pmk1Δ, and Cdc20^Slp1-DS cells exposed to 0.6 M KCl at the indicated times. Level of activated Pmk1 anti-phosho-p44/42 antibody along with Cdc2 as loading control is shown. Graph represents relative average levels of Cdc20^Slp1 in each strain from two independent experiments. Asterisk denotes p<0.001. ns: non-significant. Representative example of two independent experiments is shown. D Left, Cdc20^Slp1 levels from mitotically arrested nda3-KM311 (6.5 h) in a wild type or pmk1Δ sty1Δ backgrounds, treated or untreated with 0.6M KCl. Right, Quantitation of Cdc20^Slp1 protein levels relative to time 0 in each strain is shown. Cdc2 is used as loading control. Asterisk denotes p<0.001. ns: non-significant. Images are representative examples of three independent experiments