Fig. 5. MMS-induced Chk1 phosphorylation is abrogated in rad91-231 mutants. Indicated strains containing the cdc15-2 allele were transformed with a CHK1-HA plasmid and cultured in selective media. Cultures were arrested in telophase by incubation for 3 hours at 37°C before being mock treated or treated with 0.1% MMS (v/v) for 1 hour. Whole cell extracts were prepared and immunoblotted with -Rad9, -Rad53 or -HA antibodies. The same phosphorylation patterns were observed with two separate extracts and Chk1 phosphorylation was detected with both HA- and Myc-tagged proteins.