CK1δ-specific kinase activity in HT1080 cells is decreased after activation of Chk1.

<p>Cellular Chk1 was activated by treating HT1080 cells with 2.5 mM hydroxyurea (HU) for the indicated periods of time. (<b>A</b>) Activation of Chk1 (indicated by phosphorylated Ser-345) and expression levels of Chk1 and CK1δ were determined by immunoblotting. Detection of β-actin served as loading control. (<b>B</b>) CK1 kinase activity in fractionated extracts from HT1080 cells before and after treatment with 2.5 mM HU for 2, 4, 6 and 8 hours, respectively, was determined using GST-p53^1–64 (FP267) as substrate. The detected kinase activity was normalized towards the untreated control. (<b>C</b>) Presence of CK1δ in the kinase peak fractions shown in (B) was confirmed by use of the CK1δ-specific inhibitor compound <b>17</b> at 50 nM <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068803#pone.0068803-Peifer1" target="_blank">[82]</a>. (<b>D</b>) HT1080 cells were treated with 2.5 mM HU and/or the Chk1-specific inhibitor SB-218078 for 2 h. CK1 kinase activity in fractionated extracts was determined using GST-p53^1–64 (FP267) as substrate. The detected kinase activity was normalized towards the untreated control.