Pepstatin

Isolated hippocampal tissues were homogenized in 100 mg tissue/ml RIPA Lysis Buffer (20-188, Millipore, MA, USA). Immediately prior to use, protease inhibitor (Roche Diagnostics, Indianapolis, IN, USA) and phosphatase inhibitor (pepstatin, Roche Diagnostics, Indianapolis, IN, USA) were added into the RIPA Lysis Buffer. The resulting suspension was sonicated with an ultrasonic cell disrupter (Bandelin, OSTC) and centrifuged at 14,000× g at 4°C for 15 min. Supernatant was collected and stored at −80°C. Nuclear protein extraction was performed with NE-PER^TM Nuclear and Cytoplasmic Extraction Reagents (78833, Thermo) according to the manufacturer’s instructions, samples were stored at −80°C. Protein quantification was performed using BCA Protein Assay Kit (23227, Thermo, USA). ELISAs were performed according to the manufacturer’s instructions (IL-1β RLB00; TNF-α RTA00, R&D Systems; NF-κB ab133128; p-IκBα ab176643, Abcam; PSD-95, LS-F6865, LifeSpan BioSciences, Inc.) and optical density determined at 450 with a microplate reader (BioRad, Richmond, CA, USA).

Capsaicin (CAP), N-Acetyl cysteine (NAC) and 3-methyladenine (3-MA) were purchased to Sigma (St. Louis, USA). The inhibitors E64 and pepstatin were purchased to Roche Diagnostics (Mannheim, Germany). Primary anti-p62, anti-pAkt and antibodies were from Cell Signalling Technology (Danvers, MA, USA) and the anti-LC3 polyclonal antibody was obtained from Novus (England, UK). Peroxidase labeled secondary anti-mouse IgG was from Sigma (St. Louis, USA) and anti-rabbit IgG was from Calbiochem (San Diego, USA).

APrP was extracted using a modified water extraction protocol [29 (link)]. Three grams of cerebellar gray matter of patients 1 and 2 were homogenized in 10 ml/g TE buffer (0.1 M Tris, pH 7.4, 5 mM EDTA), and protease inhibitors (Complete, 1 mM pepstatin, 100 mM TLCK-HCl, 200 mM TPCK, and 1 mM leupeptin; all from Roche Molecular). After homogenization, 2 mM CaCl₂, 0.3 mg/ml collagenase III and 10 µg/ml DNase I (Thermo Fisher Scientific) were added. After 16 h at 37 °C, samples were centrifuged at 10,000 g for 30 min at 4 °C. The pellet was resuspended in 8 ml of 0.15 M NaCl and centrifuged at 10,000 g for 30 min at 4 °C. This step was repeated until the OD₂₈₀ of the supernatant was less than 0.075. The resulting pellet was then resuspended in 10 ml/g dH₂O and centrifuged at 30,000 g for 1 h at 4 °C. This step was repeated 5 times, and the final supernatant was centrifuged at 250,000 g for 2 h at 4 °C. The pellet was resuspended in 100 µl/g tissue in PBS with 0.5% sulfobetaine (SB3-14, Sigma), and stored at 4 °C.

Total proteins were extracted with lysis buffer (2% SDS, 10% glycerol, 63.5 mM Tris HCl, pH 6.8) containing Complete Proteinase Inhibitor (Roche Diagnostics GmbH, Vienna, Austria), 1 µg/mL pepstatin (Roche Diagnostics GmbH, Vienna, Austria) and PhosSTOP^TM phosphatase inhibitor (Roche Diagnostics GmbH, Vienna, Austria). The proteins were quantified using BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Protein extracts (50 µg) were fractionated by 10% SDS-PAGE and transferred to PVDF membranes. The blots were washed with wash buffer (PBS1x, 0.05% Twheen20), blocked for 1 h with 0.1% BSA, and incubated with primary antibodies anti phospho-myosin light chain 2 (Ser19) (dilution 1:1000, cat. No. 3671, Cell Signaling Technology, Boston, MA, USA), anti phosphoY397-FAK (dilution 1:1000, cat. No. 32835, Cell Signaling Technology, Boston, MA, USA), and anti β-actin (dilution 1:1000, cat. sc-47778, Santa Cruz Biotech, Dallas, TX, USA). The membranes were washed, and the primary antibodies were detected using HRP-coupled secondary antibodies. A chemiluminescence procedure was used for the detection of proteins (Thermo Scientific, Waltham, MA, USA).

Cells in 60 mm dishes were washed twice with phosphate-buffered saline (PBS) and lysed in situ with 0.5 ml of ice-cold CSK buffer [10 mM Pipes–NaOH (pH 6.8), 3 mM MgCl₂, 1 mM EGTA, 33.3% sucrose, 0.1% Triton X-100] containing 0.3 M NaCl, 10 mM N-ethylmaleimide (NEM; Wako Pure Chemicals), and protease inhibitor cocktail [0.25 mM phenylmethylsulfonyl fluoride (PMSF; Sigma–Aldrich), 1 μg/ml leupeptin, 2 μg/ml aprotinin, 1 μg/ml pepstatin and 50 μg/ml Pefabloc SC (AEBSF); all inhibitors, except for PMSF, were purchased from Roche Applied Science]. After incubation on ice for 1 h, the cell lysates were scraped into microfuge tubes and centrifuged for 10 min at 20 000 × g to obtain soluble extracts. The resultant pellets were resuspended with the aid of sonication in 0.25 ml of the same buffer.