Activation of Slt2 by genotoxic stress in DNA integrity checkpoint mutants. Exponentially growing cultures of the wild type (W303-1a), sml1 (JCY1144), rad53 sml1 (JCY1038), mec1 sml1 (JCY1039), mec1 tel1 sml1 (JCY1275) and tel1 (JCY1258) strains were split and incubated for 60 min in the absence or presence of 200 mM hydroxyurea or 0.04% MMS. Exponentially growing cultures of the tetO
:RAD53 (JCY1149) strain were incubated for 6 hours in the absence or presence of 5 μg/mL doxicycline in order to repress the tetO
promoter followed by 60 min incubation in the absence or presence of 200 mM hydroxyurea. The level of phosphorylated Slt2, total Slt2 protein and the chekpoint kinase Rad53 was determined by western analysis. The ponceau staining of the membranes are shown as loading control.