FIGURE 1.
Analysis of HRP::CaM6 binding to recombinant CPKs. A, spot blot overlay assay of purified His-tagged CPKs along with negative (GST) and positive (GST-GmCaMK1) controls. Nitrocellulose membranes were stained with Ponceau S to assess loading, and the same membrane was probed with 200 nm HRP::CaM6 in the presence of 2 mm CaCl2. Binding was detected as described under “Experimental Procedures.” Two different film exposures show that only CPK28 binds substantially at 200 nm HRP::CaM6. Subgroups to which each CPK belong are indicated. B, crude extracts containing His-tagged recombinant CPKs were probed for phosphoproteins by blotting with pIMAGO. Anti-His immunoblotting indicates the relative amount of each recombinant kinase. The K91E mutant of CPK28 is shown as a non-phosphorylated control. All recombinant kinases were active and autophosphorylated in situ. M, molecular weight marker; Gm, Glycine max; HRP, horseradish peroxidase; IB, immunoblot; CBB, Coomassie Brilliant Blue.