Characterization of the CK1 activity present in SV-52 and Rev2 cells.

<p><b>(A) Phosphorylation of T-Ag by CK1δKD.. </b><i>In vitro</i> kinase assays were performed using baculovirus-expressed T-Ag as a substrate and a C-terminally truncated CK1δ (CK1δKD) as enzyme. The phosphorylated proteins were separated by SDS-PAGE (12.5%) and visualized by Coomassie staining. The degree of phosphorylation was documented by autoradiography. Addition of C-terminally truncated CK1δ is indicated by + or −. kDa: kilo dalton. <b>(B) Detection of CK1 activity in fractions derived from anion exchange chromatography.</b> Soluble extracts of SV-52 and Rev2 cells were prepared and equal amounts of protein were loaded onto a 1 ml Resource Q column. The proteins were eluted with a linear gradient of increasing NaCl concentration. 0.25 ml fractions were collected, and kinase activity was determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029709#s2" target="_blank">Materials and Methods</a>. The kinase activities in the peak fractions of SV-52 and Rev2 cells were determined using either T-Ag or GST-p53^1–64 as a substrate. SV-52 cells: purple, closed circles; Rev2 cells: blue, closed triangles; — mM NaCl. <b>(C) Detection of CK1 in kinase peak fractions.</b> Western blot analyses were performed using proteins from the peak fractions of fractionated SV-52 and Rev2 cell lysates as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029709#s2" target="_blank">Materials and Methods</a>. CK1δ was detected using the CK1δ specific mouse monoclonal antibody 128A. <b>(D) Inhibition of CK1 kinase activity in SV-52 and Rev2 cellular extracts using the CK1 specific inhibitor IC261.. </b><i>In vitro</i> kinase assays were performed in the presence of 1 µM, 3 µM and 10 µM of IC261 using cellular fractions from fractionated SV-52 and Rev2 protein lysates as source of kinase. Phosphate incorporation into T-Ag and GST-p53^1–64, respectively, was normalized towards DMSO control reactions.