Ser-6 and Thr-21 are autophosphorylated by different mechanisms.

<p>Phosphorylation of GST-NtCDPK1 proteins by His-NtCDPK1 was by reacting GST-NtCDPK1 WT, GST-NtCDPK1 D219N, GST-NtCDPK1 S6A/D219N, GST-NtCDPK1 T21A/D219N, and GST-NtCDPK1 S6A/T21A/D219N with His-NtCDPK1 WT in autophosphorylation buffer. The autophosphorylation state of GST-NtCDPK1 proteins was examined using Phos-tag SDS-PAGE. GST-NtCDPK1 proteins were detected by immunoblotting with anti-GST antibody. His-NtCDPK1 WT was visualized by CBB staining. Letters represent phosphorylated GST-NtCDPK1 WT (a), unphosphorylated GST-NtCDPK1 WT, GST-NtCDPK1 S6A/D219N, and GST-NtCDPK1 S6A/T21A/D219N (b), and phosphorylated GST-NtCDPK1 D219N and GST-NtCDPK1 T21A/D219N (c). This experiment was repeated three times with similar results.