Detection of Rad53 downstream signaling by qPCR and Western blotting in <i>pph3Δ</i> cells and cells expressing various mutant alleles of <i>RAD53</i> upon MMS treatment.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037246#pone-0037246-g006" target="_blank">Fig 6A</a>. Wild-type (SJL9), <i>pph3Δ</i> (SJL7), and the various strains expressing C-terminally Myc-tagged mutant alleles of <i>RAD53</i> (HT13.1–16.1 <i>rad53-S461A</i>, <i>rad53-S461D</i>, <i>rad53-S545A</i>, <i>rad53-S545D</i> cells were incubated at 30°C in YPD containing 0.02% MMS for 4 h, then washed and recovered with fresh YPD for the indicated times. Whole cell lysates were used for immunoblot analysis with anti-Myc antibody. Untreated cells were used as control. Cdc28 was probed with anti-PSTAIRE antibody as loading control. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037246#pone-0037246-g006" target="_blank">Fig 6B</a>. Cells of wild type (S5314 or BWP17), <i>pph3Δ</i> (SJL3), <i>rad53Δ</i> (WY3), the rescued <i>RAD53</i> (HT6) and the various strains expressing mutant alleles of <i>RAD53</i> (HT13–16 <i>rad53-S461A</i>, <i>rad53-S461D</i>, <i>rad53-S545A</i>, <i>rad53-S545D</i>) were were incubated at 30°C in YPD containing 20 mM HU and 0.02% MMS for 4 h, and then recovered with fresh YPD for 6 h. RNA was extracted and reverse transcibed into cDNA for qPCR analysis. All data represent the mean of 3 independent experiments. (<i>* P</i> value<0.01; ** <i>P</i> value<0.05). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037246#pone-0037246-g006" target="_blank">Fig 6C</a>. Cells of wild type (HT1), <i>pph3</i>Δ (HT2) and the various <i>RAD53</i> mutants expressing C-terminally Myc-tagged Rfa2 (HT21–24 <i>rad53-S461A Rfa2-myc, rad53-S461D Rfa2-myc</i>, <i>rad53-S545A Rfa2-myc</i> and <i>rad53-S545D Rfa2-myc</i>) were incubated at 30°C in YPD containing 0.02% MMS for 4 h and recovered with fresh YPD over the indicated time period. Untreated cells were used as control. Total protein was extracted from harvested cells at the indicated time points and subject to immunoblot analysis with anti-Myc antibody. Cdc28 was probed with anti-PSTAIRE antibody as loading control.