P6248

Caerulein (CAE) was purchased from Sigma-Aldrich (St. Louis, MO, United States). Rabbit monoclonal antibodies against OPA1 (ab157457, Abcam Plc, Cambridge, United Kingdom), DRP1 (D6C7 rabbit mAb; Cell Signaling), LC3B [LC3B antibody (2775)]; Cell Signaling Technology, MA, United States), p62 [SQSTM1 (P-15): sc-10117; Santa Cruz Biotechnology, Santa Cruz, CA, United States)], VMP1 (D1y3E, Cell Signaling Technology, MA, United States); mouse monoclonal antibodies against Parkin (P6248, Sigma-Aldrich, St. Louis, MO, United States), V5 [13202 V5-Tag (D3H8Q) rabbit mAB-Cell Signaling)], β-actin [β-actin (C4): sc-47778; Santa Cruz Biotechnology, Santa Cruz, CA, United States)], beta tubulin (ab131205, Abcam Plc, Cambridge, United Kingdom); rabbit anti-goat antibody (Santa Cruz Biotechnology, Santa Cruz, CA, United States); goat policlonal antibodies against VDAC-1 (D-16 sc-32063; Santa Cruz Biotechnology, Santa Cruz, CA, United States); rabbit anti-mouse antibody [(315-035-048) Jackson InmunoResearch, Baltimore Pike, United States]; goat anti-rabbit antibody [(GAR):170-5046; Bio-Rad, CA, United States]. Other reagents, enzymes, and chemicals were of reagent grade and also from Sigma-Aldrich.

Control and PARK2 post-mortem brain tissues were from the Department of Neurology, Juntendo University School of Medicine. The study protocol was approved by the Human Ethics Review Committee at Juntendo University School of Medicine. Informed consent to use human tissues were obtained from patients or close family members. Patients’ data are reported in Supplementary Table 1. Control brain tissues were autopsied brain tissue from age-matched controls who had undergone neuropathological examination to exclude neurodegenerative disorders. Tissues were homogenized with lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, protease inhibitors (Roche), 50 μM MG132 and 10 mM N-ethylmaleimide (Sigma)). Western blottings were performed with Novex NuPAGE SDS–PAGE gels (Invitrogen). The following antibodies were used: GluA1 ((13185, Cell Signaling, 1:500), GluA2/3 (AB1506, Millipore, 1:1,000), GluN1 (clone N308/48, NeuroMab, 1:300), GluN2B (PA5-18536, Thermo Scientific, 1:300), GluK1 and GluK3 (H0002897 and H0002899, respectively, Abnova, 1:2,000), GluK2 (TA310550, Origene, 1:1,000), parkin (P6248, Sigma, 1:6,000), GAPDH (sc-25778, Santa Cruz, 1:1,000).

Total protein was isolated from the N2a cells using 2x SDS sample buffer (63 mM Tris-HCl, 10% glycerol, and 2% SDS). Samples (20–40 μg of protein) were electrophoresed onto a 10–15% SDS/polyacrylamide gel (SDS/PAGE) and transferred to PVDF membranes. The membranes were blocked in TBS-Tween buffer containing 20 mM Tris-HCl, 5% nonfat milk, 150 mM NaCl, and 0.05% Tween-20 (pH 7.5) for 1 hour at room temperature. Thereafter, the blot was incubated with primary parkin mouse monoclonal antibody (1 : 1000; P-6248, Sigma-Aldrich), Drp1 rabbit monoclonal antibody (1 : 1000, #8570, cell signaling), Myc-tag mouse monoclonal antibody (1 : 1000; ab18185, Abcam), and actin mouse monoclonal antibody (1 : 10,000; SC-47778, Santa Cruz) for 1-2 hours at room temperature. The membrane was washed with TBST 3 times at 10-minute intervals, incubated with the secondary antibody (1 : 5000; anti-rabbit or anti-mouse IgG conjugated with horseradish peroxidase; Jackson ImmunoResearch Laboratories) at room temperature for 1 hour, and then washed 3 times each at 10-minute interval with TBST and 2 times each for 10 minutes with TBS. Band was visualized via an enhanced chemiluminescence kit (ECL) according to the manufacturer's suggested protocol (GE Health). Membranes were then exposed to X-ray film.