<p><b>A</b>, time course of the <i>in vitro</i> kinase reaction. Rad3-Rad26 was affinity-purified from <i>S. pombe</i> using anti-Flag antibody resin, eluted with Flag peptide, and incubated with the kinase-inactive Cds1(D312E) as the substrate in the standard kinase buffer containing 200 μM ATP. At each time point, a small aliquot of the reaction was taken out and analyzed by SDS PAGE followed by Western blotting to examine the phosphorylation of Cds1-Thr<sup>11</sup>. Rad3 and Rad26 in the same samples were detected by using anti-HA and anti-flag antibody, respectively. Loading of the substrate was shown by Ponceau S staining. The kinase dead Rad3(D2249E)-Rad26 was similarly prepared and used as the control (last lane on the right). <b>B</b>, phosphorylation of Cds1-Thr<sup>11</sup> was examined in the presence of increasing concentrations of Rad3-Rad26. The reaction was carried out at 30°C for 30 min. <b>C</b>, full-length Rad4 was purified from <i>S. pombe</i>. Removal of the N-terminal GST tag was analyzed by SDS PAGE (left) and confirmed by Western blotting (right) using anti-Rad4 antibodies. Asterisks indicate the degradation products of Rad4. <b>D</b>, the Rad3 kinase reactions were carried out at 30°C for 20 min in the absence or presence of increasing amount of purified Rad4 and analyzed as in A.</p
