GCase activity assay

GCase activity was determined using a fluorometric assay with 4-methylumbelliferyl β-D-glucopyranoside (4-MU-β-Glc) as a substrate, as previously described, with certain modifications (22). All reagents were purchased from Sigma-Aldrich (Merck KGaA). After cell thawing, the fibroblasts were mixed with 200 µl 0.9% NaCl containing 1 mM phenylmethylsulfonyl fluoride, and were homogenized with an ultrasonic homogenizer (U200H; IKA Labortechnik) at 30% amplitude and 0.2% cycle. The protein concentrations of the fibroblast lysates were measured using a Bradford protein assay (Bio-Rad Laboratories, Inc.) with BSA as a standard (Bio-Rad Protein assay standard II; cat. no. 5000007, Bio-Rad Laboratories, Inc.). For the enzyme assay, 10 µl homogenized cell lysate was incubated with 90 µl 5 mM 4-MU-β-Glc in 10 mM sodium taurocholate at 37˚C for 1 h. After incubation, 200 µl 0.5 M NaHCO₃/Na₂CO₃ (pH 10.7) was added to stop the reaction. The clear reaction was transferred into a 96-well microplate, and the fluorescence emission was measured using a fluorescence spectrophotometer (SpectraMax M2/M2e Multi-Mode microplate reader; Molecular Devices, LLC) with an excitation wavelength of 365 nm and an emission wavelength of 450 nm. The GCase activity was calculated as the release of 4-methylumbelliferone (4-MU) per time divided by the quantity of protein used. One unit was defined as the release of 1 µmole 4-MU in 1 h per 1 mg protein.