In Vitro Kinase Assay for CDPK

The in vitro kinase assay was performed as previously described (Wang et al., 2019 (link)). A reaction mixture (18 μL) containing 0.40 μg of GST-CDPK-6xHis kinase, 5–10 μg of substrates of GST-fused peptides GLR3.6 or GLR3.7, and 2 μL of 10X buffer (200 mM Tris pH 7.5, 100 mM MgCl2, 10 mM EGTA, and 11 mM CaCl₂) was used. Next, 2 μL of 50 μM ATP solution (spiked with 2.5 μCi [γ-³²P] ATP) was added to initiate the kinase reaction at room temperature (24–28°C) for 10 min. The reaction was stopped by adding 5 μL of 4X sodium dodecyl sulfate (SDS) sample buffer. Samples were analyzed using 10% SDS-PAGE. The γ-³²P-labeled signals were normalized to the amount of protein determined using Coomassie brilliant blue-stained gels. The γ-³²P-labeled signals were detected using an image analyzer (GE Healthcare, Typhoon 9400).