Autophosphorylation of Ser-6 and Thr-21 leads to rapid and slow inhibition of phosphorylation of RSG, respectively.

(A) MBP-RSG was phosphorylated for the indicated time periods. Phosphorylation of Ser-114 of RSG was detected by immunoblotting using anti-pS114 antibody, which specifically recognizes phosphorylated Ser-114 of RSG. MBP-RSG was visualized by CBB staining. GST-NtCDPK1 proteins were detected by immunoblotting with anti-GST antibody. This experiment was repeated three times with similar results. (B) Statistical analysis of the data shown in (A). The quantified band intensity of phosphorylated RSG by GST-NtCDPK1 S6A/T21A at 5 min (left panel) and 60 min (right panel) was set to 1, respectively. The bar graph represents means and ± SE (n = 3). Significant differences in the phosphorylation of MBP-RSG by GST-NtCDPK1 proteins are determined by one-way ANOVA with Tukey’s honestly significant difference test at each time point. Different letters above the bars indicate significant differences between the relative intensities at each time point (P < 0.05). (C) Line graph represents the band intensities of phosphorylated RSG obtained from (A) and (B).