Purified GST-ILK is an active serine kinase.

<p>(A) Coomassie blue-stained polyacrylamide gel showing a single protein band at approximately 78 kDa, the predicted molecular weight of the GST-ILK fusion protein, prepared as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012356#s4" target="_blank"><i>Materials and Methods</i></a>. (B) Western blot analysis using antibodies specific for ILK confirm the identity of the 78 kDa band. (C) Analysis of the concentration dependence of the protein kinase activity of GST-ILK using GSK-3 crosstide and LC<sub>20</sub> as substrates. Experiments were carried out using standard kinase reaction conditions (described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012356#s4" target="_blank"><i>Materials and Methods</i></a>) in the presence of 10 mM MgCl<sub>2</sub>. Representative autoradiographs (upper panels) show the amount of <sup>32</sup>P-phosphate incorporation with increasing concentrations of GST-ILK. Coomassie stains of SDS-gels (lower panels) depict equal loading of substrate into the kinase reaction. (D) Time course of the protein kinase activity of GST-ILK. Reactions were performed as described in C using GSK3 crosstide and LC<sub>20</sub> as substrates. The amount of ILK was held constant at 30 ng. (E) Phosphorylation of Ser9 on GSK3 crosstide and Ser19 on LC<sub>20</sub>. Each substrate was incubated with 150 ng of GST-ILK in 25 µl of kinase reaction buffer (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012356#s4" target="_blank"><i>Materials and Methods</i></a>) for the indicated times in the presence of 500 µM cold ATP. Western blot analysis was carried out using phospho-specific antibodies as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012356#s4" target="_blank"><i>Materials and Methods</i></a>. Ponceau S stains of membranes are provided as loading controls.</p>

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