File:Fpls-02-00036-g003.png

English: Kinase reactions showing Ca2+-dependent phosphorylation of a Di19-site-S109 fusion protein (G-GFP-S109-ST) by CPK16. Three kinase reactions are shown on the left side after separation on an SDS-12% polyacrylamide gel and staining with Coomassie Brilliant Blue (1s,2s, 3s). The same gel is shown to the right as an autoradiogram to show the 32P-labeling of phosphorylated proteins (1r, 2r, 3r). Kinase reactions were conducted as described in Section “Materials and Methods,” plus (+) or minus (−) Ca2+, with 1 μg of fusion protein substrate, 0.01 μg of kinase, in a 10-μL reaction. After a 10-min incubation, the entire reaction was subjected to SDS-PAGE. In control reactions, CPK16 did not phosphorylate the GST–GFP portion of the Di19-2-site-S109 fusion protein (e.g., see Figure 1). Note that the purified G-GFP-S109-ST fusion protein runs as two bands with apparent molecular weights of 48 and 60 kD. Results shown are representative of multiple independent analyses.