Affinity purified antibody to yeast RAD9 ( 1125-1139 pan reactive ) was used at a 1:200 dilution incubated 8 h at room temperature to detect RAD9 by Western blot. Lanes were loaded with 50 ng each of recombinant GST fusion protein containing S. cerevisiae RAD9 ( aa 991-1309 ~60 kDa ) on a 4-20% Criterion gel for SDS-PAGE as follows: Lane 1 - non-phosphorylated wild type yeast RAD9, Lane 2 - in vitro phosphorylated wild type yeast RAD9, Lane 3 - non-phosphorylated S1129A/S1260A double mutant RAD9, Lane 4 - in vitro phosphorylated S1129A/S1260A double mutant. Phosphorylation of RAD9 was by treatment with ATP and Rad53 kinase.