In vitro Phosphorylation Assay

The in vitro kinase assay was performed according to previously described method with small modification (Hashimoto et al., 2012). Briefly, StrepII-tagged ZmCBL1, ZmCBL9, and ZmCIPK23 proteins were generated using a RTS 100 wheat germ CECF kit and purified with Strep-Tactin Macroprep (IBA). GST tagged ZMK1-C protein was expressed in E. coli. and purified with Glutathione Sepharose 4B (GE Healthcare). About 1 μg ZMK1-C protein and 0.2 μg ZmCBL1, ZmCBL9, and ZmCIPK23 proteins were used in the phosphorylation reaction. Purified proteins were incubated for 30 min at 30°C in 30 μl reactions containing 20 mM Tris (pH 8.0), 1 mm MnSO₄, 0.5 mM CaCl₂, 1 mM DTT, 10 μM ATP, and 3 μCi of [γ-32P]ATP. The reactions were terminated by adding 6 μl 6×SDS protein sampling buffer and then subjected to SDS-PAGE. SDS gels were stained with Coomassie blue, and radioactively labeled proteins were visualized by autoradiography.