DOX and ETOP induce activation of Chk1 and Chk2 kinases and inhibition of Cdc2 kinase.

<p>(A) MCF-7 Cells were incubated with 1 µM DOX (upper panel) or 10 µM ETOP (lower panel) for the indicated times for up to 2 hr (lanes 1–4). For the 4 hr and 6 hr time points (lanes 5–6), the cells were incubated for 2 hr with DOX or ETOP, washed with DMEM and incubated for additional 2 hr and 4 hr, respectively, in regular culture medium. Following treatment, Chk1 and Chk2 kinases were respectively immunoprecipitated from cell lysates and examined for kinase activity as described in <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050281#s2" target="_blank">Materials and Methods</a></i> (<i>Chk1 Activity</i> and <i>Chk2 Activity</i>). Levels of Chk1 and Chk2 in the immunoprecipitates were determined by immunoblotting (<i>Chk1 IP-WB</i> and <i>Chk2 IP-WB</i>). *, as a negative control, kinase assay was carried out using immunoprecipitates obtained by incubating DOX-treated cell sample (6 hr time point) with non-immunized IgG. (B) Cells were treated as described above and incubated for 2 hr. Cdc2 was immunoprecipitated from cell lysate and analyzed for levels of Cdc2-Tyr15 phosphorylation by immunoblotting (<i>Cdc2-Tyr15</i>). As a control, Cdc2 in the immunoprecipitates was assessed by immunoblotting (<i>Cdc2</i>).