Figure 6.

In vitro phosphorylation of OsAP37 by GUDK. A, GST pull-down assay showing the interaction of GUDK with OsAP37. The OsAP37-6xHis fusion protein was incubated with GUDK-GST fusion protein on Glutathione Sepharose 4B resin and the protein-protein interaction was detected by immunoblot analysis using His-antibody (1:1,000 dilution). The purified pGEX:GST (vector) induced with 1 mm isopropylthio-β-galactoside was used as the control. B, In-solution kinase assay showing the phosphorylation of OsAP37 by GUDK. The assay was performed with GUDK-GST fusion protein and OsAP37-6xHis fusion protein using the Kinase-Glo Luminescent Kinase Assay Platform and the kinase activity was expressed as the reciprocal of the luminescence signal. A control assay was run without OsAP37 and GUDK. Each bar represents the means ± se of three independent experiments. C, In vitro phosphorylation assay conducted using GUDK-GST fusion protein and OsAP37-6xHis fusion protein. The interaction was detected by immunoblot analysis using an phospho-Tyr antibody. [See online article for color version of this figure.]