Phosphorylation site identifcaiton of OsHSFA4d

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.

为鉴定被OsCDPK24磷酸化的OsHSFA4d氨基酸残基，本研究通过孵育重组MBP-HA-OsCDPK24与GST-OsHSFA4d的混合体系、以及MBP-HA与GST-OsHSFA4d的混合体系，开展了体外激酶活性测定实验。通过添加终浓度为1×的SDS上样缓冲液终止反应，随后按照前述方法对反应产物进行考马斯亮蓝（CBB）染色与免疫印迹分析。切下对应磷酸化OsHSFA4d的考马斯亮蓝染色蛋白条带，随后进行液相色谱-串联质谱（LC-MS/MS）分析。