Figure 1. Purified GST-ILK is an active serine kinase.

(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 Materials and Methods . (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 LC₂₀ as substrates. Experiments were carried out using standard kinase reaction conditions (described in Materials and Methods ) in the presence of 10 mM MgCl₂. Representative autoradiographs (upper panels) show the amount of ³²P-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 LC₂₀ as substrates. The amount of ILK was held constant at 30 ng. (E) Phosphorylation of Ser9 on GSK3 crosstide and Ser19 on LC₂₀. Each substrate was incubated with 150 ng of GST-ILK in 25 µl of kinase reaction buffer (see Materials and Methods ) 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 Materials and Methods . Ponceau S stains of membranes are provided as loading controls.