Aprotinin

RAW264.7 cells were seeded at 2.0 × 10⁵ cells/well in 6-well plates and incubated for 24 h at 37°C under 5% CO₂. Then, cells were washed with PBS, mechanically detached by pipetting. Mouse lung tissue were obtained from male ICR mice (SLC, Hamamatsu, Japan). Lung tissue were disrupted by the ultrasonic disrupter (TOMY, Tokyo, Japan). The cells and lung tissue were lysed in radio-immunoprecipitation assay buffer containing protease inhibitors (Antipain; #4062 and Leupeptin; #4041, PEPTIDE INSTITUTE INC., Osaka, Japan, Aprotinin; #033–46, nacalai tesque, Kyoto, Japan, concentration of each reagents; 2.0 μg/mL) and phosphatase inhibitor (4906837001, Sigma-Aldrich) for 30 min on ice. The supernatant of the resulting suspension was obtained after centrifugation (16,000 × g, 20 min, 4°C) and collected as the total cell lysate. The total protein concentration was quantified using a Bradford protein assay kit. Animal experiments were approved by the institutional guidelines of the Centre for Experimental Animal Care and Use, Kindai University Faculty of Medicine (KAME-26-040).

Brain samples were obtained from Sv2a^L174Q or F344 rats under pentobarbital (80 mg/kg, i.p.) anesthesia and chilled in ice-cold saline. Brains were then dissected into various region blocks and homogenized in an ice-cold lysis buffer (pH 7.5) containing: (in mM) Tris 20, NaCl 150, MgCl₂ 10, EDTA 1.0, 1% Triton X-100 and a mixture of protease inhibitors (leupeptin, aprotinin, E-64, pepstatin A, bestatin and 4-(2-Aminoethyl) benzensulfonyl fluoride hydrochloride) (Nakarai Tesque, Tokyo, Japan). The homogenate was centrifuged at 15,000 g, 4 °C for 30 min and the supernatant was stored at −80 °C for subsequent analysis.
Western blotting was performed according to the methods described previously14 (link)23 (link)24 (link), using the corresponding primary antibodies as follows, mouse monoclonal antibody against Stxbp1 (1:2000, Synaptic Systems), Nsf (1:2000, Synaptic Systems), Napa (1:2000, Santa Cruz Biotechnology), Syt1 (1:5000, Synaptic Systems) and β-actin (dilution 1:2000, Sigma Aldrich, St Louis, MO) and a sheep anti-mouse IgG-HRP conjugate (dilution 1:2000, GE Healthcare) as the secondary antibodies. Final detection was performed with the enhanced chemiluminescence method (Amersham ECL Western blotting detection reagents and analysis system, GE healthcare, Buckinghamshire, UK) using a lumino imaging analyzer (LAS-3000, FUJIFILM, Tokyo, Japan).

Sol, Pla, and Gas samples were homogenized in lysis buffer (50 mmol/L Hepes, pH 7.4, 150 mmol/L NaCl, 2 mmol/L EDTA, 1% sodium deoxycholate, 1% NP-40, and 0.2% sodium dodecyl sulfate) with a protease inhibitor mixture (AEBSF, aprotinin, E-64, leupeptin, bestatin, and pepstatin A; Nacalai Tesque Inc., Kyoto, Japan) on ice. Protein concentrations were measured using a Protein Assay Bicinchoninate Kit (Nacalai Tesque Inc.).