Fig. 7

OsANN4 can interact with and be phosphorylated by the protein kinase OsCDPK24. a A yeast two-hybrid system was used to detect the interaction between OsANN4 and OsCDPK24. Yeast cells containing the indicated constructs were grown on dropout medium without Leu and Trp (-LW, right panel) and without Leu, Trp, His and Ade (-LWHA, left panel). b A GST pull-down assay was used to detect the interaction between OsANN4 and OsCDPK24. OsCDPK24-His protein was incubated with OsANN4-GST or GST protein. Pull-down proteins were detected by GST-tagged and His-tagged antibodies. c Firefly luciferase complementation imaging (LCI) assay detecting the interaction between OsANN4 and OsCDPK24. The colored scale bar indicates the luminescence intensity. d An in vitro kinase assay was performed with purified OsANN4-His and OsCDPK24-His using an SDS-PAGE gel (8%) containing a 50 μM Phos-tag. Signals were detected by using a His-tag antibody. e Mass spectrometry analysis of OsANN4 phosphorylation