Recombinant human MLK3 active protein (no. M19-11G, SignalChem) was heat-inactivated at 65°C for 20 min in a sonicating bath. To confirm heat inactivation of MLK3 recombinant protein, kinase reactions between MLK3 heat-inactive protein (1 μg) and myelin basic protein substrate (5 μg; no. M42-51N, SignalChem) were performed. To determine whether ULK1 can directly phosphorylate MLK3, kinase reactions between recombinant human ULK1 active protein (100 ng; no. U01-11G, SignalChem) and MLK3 heat-inactive protein (1.66 μg) were performed. As controls, the same kinase reactions were carried out, but using each recombinant protein alone. For each kinase reaction, we used dithiothreitol (no. D86-09B, SignalChem), ATP (no. V915A, Promega), and 5× kinase buffer (no. K03-09, SignalChem), following the manufacturer’s instructions. Kinase mixtures were incubated for 40 min shaking at 300 rpm and 30°C. The ADP formed from the kinase reactions was measured using the ADP-Glo Kinase Assay Kit (no. V6930, Promega), following the manufacturer’s instructions.

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