Phosphorylation of Mec1 and Mec1/Tel1 substrates in <i>rad53</i> mutant cells.

<p>TCA-extracted protein samples were taken from untreated cells (U) or 90 minutes MMS-treated cells (0.02% MMS except where indicated) (M), processed for Western blotting after SDS-PAGE in appropriate gels (from 8% to 12%), and probed with α-γH2AX (ab15083, Abcam), α-myc (M^.5546, Sigma-Aldrich), α-Ha (12CA5, Sigma-Aldrich) or α-Rad53 (sc-6749, Santa Cruz Biotechnology Inc.) antibodies to detect histone H2A phosphorylated at S129, myc-tagged Slx4, Ha-tagged Ddc1 and Rad53, respectively. (A) γH2AX antibody immunoreactivity of whole cell extracts from wild-type and <i>rad53Ha</i> (r53Ha), <i>rad53^S350A,G404V</i> (r53^350,404) and <i>rad53Δ</i> (r53<i>Δ</i>) mutants untreated (U) or MMS-treated (M) as indicated. (B) (Upper panel) γH2AX antibody immunoreactivity and (middle panel) myc antibody immunoreactivity of whole cell extracts from <i>slx4-myc-</i>tagged cells, otherwise wild-type (wt), <i>rad53Ha</i> (r53Ha), <i>rad53Δ</i> (r53<i>Δ</i>) or <i>mec1Δ</i>, untreated (U) or MMS-treated (M). (C) Detection of γH2A (upper blot), Ddc1-Ha and Rad53 (lower blots) in whole cell extracts from <i>ddc1-Ha-</i>tagged cells, otherwise wild-type (wt), <i>mec1-1</i>, <i>rad53Ha</i> (r53Ha) or <i>rad53Δ</i> (r53<i>Δ</i>), untreated (U) or MMS-treated (M).