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

Figure 2.

Figure 2

Wild-type Hsk1 but not kinase-negative or kinase-attenuated Hsk1 restores HU resistance or hyperphosphorylation of Mrc1 in hsk1-89. pREP 81 (Vec), pREP 81-Hsk1 wild-type (Hsk1wt), pREP 81-Hsk1 K129D (Hsk1-kd; kinase-dead) or pREP 81-Hsk1 KK129,130RS[KK-RS] (Hsk1-ka; kinase-attenuated) was transformed into hsk1+ (YM71) or hsk1-89 (KO147) cells. (A) Five-fold serial dilutions of exponentially growing cells harboring each plasmid indicated were plated on EMM supplemented with Uracil (EMMura) agar medium with or without thiamine and incubated at 25 or 30°C for seven days as indicated. One set of plates (right part for each temperature) contained 8 mM HU. (B and C) The same set of cells were grown at 25°C in EMMura with or without thiamine as indicated. For HU-treatment at 25°C, 15 mM HU was added to the half of the culture and the other half was non-treated. The incubation was continued for 4 hrs at 25°C. For HU-treatment at 30°C, 25 mM HU was added to the half of the culture and the other half was non-treated. The incubation was continued for 3 hrs at 30°C. The whole cell extracts were prepared and analyzed by western blotting to detect Mrc1 and Hsk1 proteins.