Cell lysate preparation

SH-SY5Y cells were harvested by trypsin-EDTA 0.25% (Thermo Fisher Scientific, Inc.) and collected by centrifugation at 1,000-2,000 x g for 10 min at 4˚C. Cells were then harvested in ice-cold buffer (0.05 M potassium phosphate pH 7.0, 1 mM EDTA) and homogenized by sonication on ice. The solution was centrifuged at 12,000 x g at 4˚C for 20 min to remove cell debris. The supernatant was used for determining the quantity of total protein and for the enzyme activity assay. The protein concentration was determined using the bicinchoninic acid assay kit (Thermo Fisher Scientific, Inc). All spectrophotometric measurements were made using an Epoch microplate spectrophotometer (BioTek Instruments, Inc.).