AMPK enzymatic activity assay

Recombinant AMPK activity was assessed using a modified non-radioactive approach as published previously [9]. Briefly, recombinant-AMPK α2β1γ1 (400 ng/mL, Invitrogen) was pre-incubated with QVO (20 μM) or A-769662 (1 μM) for 30 min on ice. The kinase reaction was initiated upon adding ATP (50 μM) and substrate SAMS (100 μM; Sangong, Shanghai, China) at room temperature for 30 min, avoiding light. The generation of ADP reflected AMPK enzyme activity, and ADP was detected by the ADP Hunter Plus assay kit (Discover X, CA, USA). The fluorescent signal was detected using an M5 multi-detection reader (Molecular Devices, CA, USA) at an excitation wavelength of 530 nm and an emission wavelength of 590 nm.