In vitro GST pull‐down assays

The GST pull‐down assays were performed as previously described. Recombinant proteins, 10 μg GST or GST‐P1 and the indicated amount of MBP‐OsCIPK23 were incubated in binding buffer [25 mm Tris–HCl, pH 7.5, 100 mm NaCl, 10 mm MgCl₂, 1 mm DTT, and 10% (v/v) glycerol] containing 40 μL of GST beads and incubated at 4 °C overnight with continuous slow shaking. After incubation, the combined GST resin was subjected to wash three times with a washing buffer [25 mm Tris–HCl, pH 7.5, 250 mm NaCl, 10 mm MgCl₂, 1 mm DTT, 10% (v/v) glycerol, and 0.1% Triton X‐100]. This aimed to remove nonspecific proteins that may have bound to the resin. The proteins bound to the GST resin were eluted using an elution buffer [25 mm Tris–HCl, pH 7.5, 20 mm glutathione, 100 mm NaCl, 10 mm MgCl₂, 1 mm DTT, and 10% (v/v) glycerol]. The eluted proteins were separated on a 12% SDS‐polyacrylamide gel and subjected to immunoblotting analysis using an anti‐MBP antibody. The input proteins were also separated on a 12% SDS‐polyacrylamide gel and assessed using immunoblot analysis to confirm their presence in the assays.