Rad53 activation in the
Western blots of crude protein lysates of mcm2AA or MCM2 strains probed with anti-Rad53 antibody before and after treatment with MMS. Log phase cultures in YPD were grown for 2 hours at 30°C in the presence or absence of 0.02% MMS. After harvesting by centrifugation, protein was extracted with TCA, as described . Approximately 20 μg of total protein was examined by Western blotting using anti-Rad53 antibody (Santa Cruz Biotechnology, SC-6749) as primary antibody followed by rabbit anti-goat IgG coupled to horseradish peroxidase (Sigma-Aldrich). The blots were visualised using the Supersignal West Pico chemiluminescence kit (Pierce) and x-ray film.