FIGURE 2.

Interaction between CPK28 and CaM is Ca²⁺-dependent. A, spot blot overlay assay of purified His₆-CPK28 probed in the presence (2 mm CaCl₂) or absence (5 mm EGTA) of Ca²⁺ or in the presence of Ca²⁺ with addition of the high affinity CaM-binding peptide W3 (2 mm CaCl₂ + W3). B, binding of CPK28 to immobilized CaM (CaM-Sepharose). 20 μg of purified His₆-CPK28 was bound to immobilized CaM in the presence of Ca²⁺ (2 mm CaCl₂) with (+W3) or without (−W3) the addition of the W3 peptide. After washing three times with buffer containing 2 mm CaCl₂, bound proteins were eluted in three fractions with buffer containing 5 mm EGTA. Fractions were assessed by immunoblotting with anti-His₆ antibodies.