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. 2010 Dec 1;9(23):4627–4637. doi: 10.4161/cc.9.23.13937

Figure 4.

Figure 4

Rad3 or Hsk1 kinase activity is not affected in hsk1-89 or rad3Δ cells, respectively. Kinase assays were conducted as described in “Experimental Procedures” with anti-Myc antibody (A) or anti-Flag antibody (B) immunoprecipitates. (A) Extracts were prepared from non-tagged (lanes 2 and 3, YM71), Myc-tagged Rad3 (lanes 4 and 5, SH5142) and Myc-tagged Rad3 hsk1-89 (lanes 6 and 7, SH5431) strains. Cells were grown at 25°C and 12 mM HU was added (lanes 3, 5 and 7) or non-treated (lanes 2, 4 and 6), and incubation was continued for 1.5 hrs at 30°C. Lane 1, reaction without kinase. The reactions were conducted in the presence of 8 mM magnesium acetate, and thus the background phosphorylation of Mrc1 in non-tagged extract is observed. The shifted band, representing Rad3-mediated phosphorylation, is generated only by the tagged extract. Upper, autoradiogram; middle, silver staining; lower, immunoblot with anti-Myc antibody. (B) Extracts were prepared from non-tagged (lanes 1–4) or Flag-tagged Hsk1 (lanes 5–8) strain. Lanes 1, 2, 5 and 6, rad3+: lanes 3, 4, 7 and 8; rad3Δ. Cells were grown at 30°C and 12 mM HU was added (lanes 2, 4, 6 and 8) or non-treated (lanes 1, 3, 5 and 7), followed by further incubation for 1.5 hrs. Lane 9, reaction with purified Hsk1-Dfp1/Him1 complex; lane 10, reaction without kinase. Lanes 1 and 2, YM71; lanes 3 and 4, NI392; lanes 5 and 6, MS337; lanes 7 and 8, MS420. Upper, autoradiogram; lower, silver staining.