ADP-Glo kinase assay

An ADP-Glo kinase assay kit (Promega) was used to measure the kinase activity of the ULK1 complex. Briefly, 100 nM purified wild-type or KD ULK1 complex was mixed with 5 μM ULKtide (SignalChem Biotech Inc.) in the presence or absence of 100 μM ATP in a reaction buffer containing 20 mM Hepes (pH 8.0), 200 mM NaCl, 2 mM MgCl₂, 20 mM maltose, and 1 mM TCEP. The reaction was performed at room temperature for 1 hour and terminated by an ATP depletion reagent. Then, the ADP was converted to ATP by a kinase detection reagent, which was used in a coupled luciferase reaction. The luminescent output was measured with a GloMax-Multi detection system (Promega) and correlated with the kinase activity.