File:Fpls-02-00036-g004.png

English: Kinase reactions with CPK16 comparing relative phospho-labeling of Di19-2-site-S109 and variants. (A) For each amino acid substitution, the fold change in phosphorylation is shown in comparison to phosphorylation levels for the wild type version of the S109 site encoded in G-GFP-S109-ST. All substitutions were engineered by site-specific mutagenesis of the same parental clone, G-GFP-S109-ST. All reactions, SDS-PAGE gels, and autoradiograms were performed as described in Figure 2 and in Section “Materials and Methods.” The percentage incorporation of 32P was determined by analysis of autoradiograms and was normalized to the amount of Coomassie Brilliant Blue-stained protein for each substrate. Results shown are the average of at least four independent analyses for each variant. Error bars show SD. (B) A summary of changes in phosphorylation as a result of substitutions. Peptides with similar phosphorylation potentials are shown first (i.e., changes less than twofold compared to wild type), followed by those that were higher or lower by more than twofold.