Analysis of the hypersensitivity of slt2 cells to genotoxic stress and the activation of Slt2 by genotoxic stress in osmotically supported medium. A) 10-fold serial dilutions from exponentially growing cultures of wild type (W303-1a) and slt2 (JCY1062) strains were spotted onto YPD medium containing 1 M sorbitol and 200 mM hydroxyurea or 0.03% MMS or were exposed to UV radiation (40 J/m2). Plates were incubated at 25°C for 3 days. B) Cultures of the wild type W303-1a strain grown on YPD containing 1 M sorbitol were split and incubated for 60 min in the absence or presence of 200 mM hydroxyurea, 0.04% MMS or 5 μg/mL phleomycin, or were irradiated with UV radiation (50 J/m2). The level of phosphorylated Slt2 and the chekpoint kinase Rad53 (as a control of the presence of genotoxic stress) was determined by western analysis. The ponceau staining of the membrane is shown as loading control.