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Glucocerebrosidase assay

EB Electra Brunialti
AV Alessandro Villa
MM Marianna Mekhaeil
FM Federica Mornata
EV Elisabetta Vegeto
AM Adriana Maggi
DM Donato A. Di Monte
PC Paolo Ciana
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A glucocerebrosidase assay was performed as previously described [21]. Cells were lysed with RIPA buffer, and the protein concentration was determined with BCA (Cat. 23227, Pierce). The biochemical assay of GCase activity was carried out in a buffer containing 4-methylumbelliferyl beta-d-glucopyranoside substrate (Cat. M3633, Sigma-Aldrich); after 1 h at 37 °C, the reaction was stopped with 0.25 M glycine buffer (pH 10.4), and the fluorescence emission of the 4-methylumbelliferyl generated by the reaction was read with a fluorimeter (EnSpire Plate Reader, PerkinElmer) with an excitation wavelength of 365 nm and an emission wavelength of 445 nm. The determined enzyme activity was expressed as the μmol 4-methylumbelliferyl generated in 1 h per μg of protein and normalized to the value in vehicle-treated cells.

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