Fig. 2. Phosphorylation of Rad9, Hus1 and Rad26 in response to DNA damage and/or inhibition of DNA replication is independent of Rad4. Wild-type and rad4-116 cells carrying integrated, epitope-tagged alleles of hus1, rad9 or rad26 were used in these experiments. Cells were grown overnight to mid-log, harvested and inoculated into fresh pre-warmed media in the absence (-) or presence (+) of 20 µg/ml bleomycin sulphate; (A) Hus1-myc, (B) Rad9-HA, (C) Rad26-myc. (D) Exponentially growing Hus1-myc cells were treated with 10 mM HU for 5 hours. Total protein was prepared from all cells and phosphorylation analysed by immunoblot using either anti-HA or anti-myc antibodies.