Figure 8

Analysis of Swe1 protein level in slt2 mutant cells after a replicative stress. A) Exponentially growing cells of the wild type (JCY1316) and slt2 (JCY1318) strains expressing a HA-epitope tagged Swe1 protein were incubated in the absence or presence of 200 mM hydroxyurea. Swe1 protein level and activation of the chekpoint kinase Rad53 was analyzed at the indicated time after the addition of HU by western blot. A non-specific band labelled with an asterisk that cross-react with the antibody is shown as loading control. Graph represents the relative amount of Swe1 protein related to the non-specific band in the HU-treated cells derived from three independent assays. B) Exponentially growing cultures of the slt2 (JCY1062) and the slt2 swe1 (JCY1633) strains were incubated in the presence of 200 mM hydroxyurea for 6 hours. Cell cycle distribution of cells and the presence of abnormal elongated bud were analyzed as described in Figure 7. Graphs represent cell distribution derived from at least three independent cultures. C) Aliquots from exponentially growing cultures of the swe1 (JCY1632) and the slt2 swe1 (JCY1633) strains were incubated for 90 min. at the indicated doses of HU, MMS and phleomycin or were exposed to different doses of UV radiation. Cells were plated on YPD and the percentage of surviving cells relative to untreated controls was determined.