In Vitro Phosphorylation Assay of Atg Proteins

The GST-MoMkk1, His-MoAtg1, His-MoAtg9, His-MoAtg9^6A, His-MoAtg9^5A were expressed in Escherichia coli BL21 cells. In vitro, the rapid and cost-effective fluorescence detection in tube (FDIT) method was used to analyze protein phosphorylation with the Pro-Q Diamond Phosphorylation Gel Stain (Thermo Fisher Scientific, P33301), a widely used phosphor-protein gel-staining fluorescence dye. For protein kinase reaction, 0.2 µg MoMkk1 (MoAtg1) was mixed with 2 µg MoAtg9 or MoAtg9^6A (MoAtg9^5A), in a kinase reaction buffer (100 mM PBS, pH 7.5, 10 mM MgCl₂, 1 mM ascorbic acid, with the appearance of 50 µM ATP) at room temperature (RT) for 60 min, followed by 10-fold of cold acetone was added to stop the reaction. Then, the protein was precipitated in a −20°C freezer for 4 h and centrifuged at 13,200 g for 1 h at 4°C. Phosphorylation protein was stained by 100 µL of Pro-Q Diamond Phosphorylation Gel Stain (Thermo Fisher Scientific, P33301) and kept in the dark at RT for 1 h. Then, the sample was added 10-fold of cold acetone and precipitated in a −20°C freezer for 4 h and centrifuged at 13,200 g for 1 h at 4°C again. The protein was washed with 0.5 mL cold acetone twice and dissolved in 200 µL of Mili-Q water after air-drying. The fluorescence signal was measured in a Cytation3 microplate reader (Biotek, Winooski, VT, USA) at 590 nm (excited at 530 nm) (68 (link)).