Aprotinin

The AChE assay was performed using an AChE assay kit (Abcam, Cambridge, UK) according to the manufacturer’s protocols. Briefly, the hippocampus from the brain of each mouse was homogenized in PRO-PREP protein extraction solution (1.0 mM PMSF, 1.0 mM EDTA, 1.0 μM pepstatin, 1.0 μM leupeptin, and 1.0 μM aprotinin, iNtRON Biotechnology, Inc., Seoul, Korea), after which the homogenates were stored at −70 °C until analysis. The samples or standards and ACh reaction mixtures were then incubated on a 96-well plate for 20 min at room temperature while protected from the light. Color alterations were read using a Vmax plate reader (Molecular Devices, Sunnyvale, CA, USA) at 405 nm.

Cell lysates or tissue strip lysates were prepared on ice in PRO-PREP lysis buffer (10 mM HEPES, pH 7.9, 10 mM KCl, 2 mM MgCl₂, 0.5 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 μg/mL aprotinin, 1 μg/mL pepstatin A, and 2 μg/mL leupeptin; iNtRON Biotechnology, Gyeonggi-do, Korea). Protein extracts (20~30 μg) were separated on 12–15% SDS-PAGE and analyzed via Western blot followed by an enhanced chemiluminescence system (ECL, Amersham Bioscience, Piscataway, NJ, USA). The following working dilutions were used: anti-TH (1:1000), anti-AKT (1:1000), and anti-pAKT (1:1000). HRP-conjugated secondary antibodies were diluted to 1:3000 for anti-mouse IgG and 1:2000 for anti-rabbit IgG. Rabbit polyclonal antibodies against human TFAM (1:2000) were prepared in our laboratory and used for TFAM detection^{49 (link)}. Anti-β-actin antibody (1:3000) was used as a loading control.

The level of AChE activity was determined by using an Acetylcholinesterase Assay Kit (Abcam, Cambridge, UK) in accord with the manufacturer’s protocols. Briefly, the brain cortex from each mouse was homogenized in PRO-PREP protein extraction solution (1.0 mM PMSF, 1.0 mM EDTA, 1.0 μM pepstatin, 1.0 μM leupeptin, and 1.0 μM aprotinin; iNtRON Biotechnology Inc., Seoul, Korea); prior to AChE analysis, the homogenate was stored at −70 °C. For analysis, the homogenate sample (or standards) and the AChE reaction mixture were incubated for 20 min at room temperature in a 96-well plate protected from light. Color alteration within the plate wells was determined by using a Vmax plate reader (Molecular Devices, Sunnyvale, CA, USA) at 405 nm.