To identify the residues of OsHSFA4d phosphorylated by OsCDPK24, the in vitro kinase assays were conducted by incubating recombinant MBP-HA-OsCDPK24 + GST-OsHSFA4d and MBP-HA + GST-OsHSFA4d. The reaction was terminated by adding SDS loading buffer to a 1×concentration, and the reaction products were subjected to CBB staining and immunoblot analysis as described above. The CBB-stained protein bands corresponding to phosphorylated OsHSFA4d were excised and subjected to LC-MS/MS analysis.