Felix software

β- and γ-secretase activity in mice brains was determined using a commercially available β- and γ-secretase activity kit (Abcam, Inc, Cambridge, MA, USA). Protein was extracted from brain tissues (hippocampus regions) using protein extraction buffer (PRO-PREP™, Intron Biotechnology, Korea), incubated on ice for 1 h, and centrifuged at 13,000g for 15 min at 4 °C. The supernatant was collected. A total of 50 μl of sample (total protein 100 μg) was added to each well followed by 50 μl of ×2 reaction buffer and 2 μl of β-secretase substrate incubated in the dark at 37 °C for 2 h. Fluorescence was read at excitation and emission wavelengths of 355 and 510 nm, respectively, using a Fluostar Galaxy fluorimeter (BMG Lab Technologies, Offenburg, Germany) with Felix software (BMG Lab Technologies, Offenburg, Germany). β- and γ-secretase activity is proportional to the fluorimetric reaction and is expressed as nanomole per milligram of protein per minute.

β-Secretase activity in all mice brains was determined using a commercially available β-secretase activity kit (R&D Systems, Minneapolis, MN, USA) and a BACE1 fluorescence resonance energy transfer assay kit (Panvera, Madison, WI, USA). Fluorescence was read at excitation and emission wavelengths of 355 and 510 nm, respectively, using a Fluostar Galaxy fluorimeter (BMG Lab Technologies, Offenburg, Germany) with Felix software (BMG Lab Technologies, Offenburg, Germany). β-Secretase activity is proportional to the fluorimetric reaction and is expressed as nanomoles per milligram protein per minute. γ-secretase activity was performed in ex vitro using CHAPSO-solubilized membrane fractions. Enzyme activity levels were quantified based on the description by Farmery et al [53 (link)].

β-Secretase activity in mice brains was determined using a commercially available β-secretase activity kit (Abcam, Inc, Cambridge, MA, USA). Protein was extracted from brain tissues (hippocampus regions) using ice-cold extraction buffer, incubated on ice for 20 min and centrifuged at 10,000×g for 5 min at 4 °C. The supernatant was collected. A total of 50 μl of sample (total protein 100 μg) was added to each well followed by 50 μl of 2 × reaction buffer and 2 μl of beta-secretase substrate incubated in the dark at 37 °C for 2 h. Fluorescence was read at excitation and emission wavelengths of 355 and 510 nm, respectively, using a Fluostar Galaxy fluorimeter (BMG Lab Technologies, Offenburg, Germany) with Felix software (BMG Lab Technologies, Offenburg, Germany). Beta-secretase activity is proportional to the fluorimetric reaction and is expressed as nanomole per milligram of protein per minute.