Sodium dodecyl sulfate (sds)

Six (n = 5) and 24 h (n = 6) after SCI with 30 g/mm² of clipping power for a duration of 15 s, the spinal cord was dissected into rostral, epicentre, and caudal segments, each of which was 2 mm in length. Each spinal cord segment was minced with a homogenizer in RIPA buffer containing 50-mM Tris–HCL 7.5 (Nippon Gene Co., Ltd), 150-mM NaCl (Nacalai Tesque), 0.1% SDS (Nippon Gene Co., Ltd), 1% NP-40 (Sigma-Aldrich), 5-mM EDTA with pH 8.0 (Nippon Gene Co., Ltd ), and 1 mM PMSF (Wako). Protease inhibitor cocktail (Thermo Fisher Scientific) was added to the buffer prior to homogenization in accordance with the manufacturer’s instructions (1:100). The spinal cord was homogenized for ∼40 grinds using constant force to ensure consistency and homogeneity of the samples. The homogenates were divided into roughly equal volumes in Eppendorf tubes and stored at 4°C for 30 min. The homogenates were then centrifuged at 13 000 rpm for 20 min at 4°C. The resulting supernatant was decanted, placed into newly labelled Eppendorf tubes, and immediately stored at −80°C. The protein concentration was determined using Qubit (Thermo Fisher Scientific). Equal amounts of protein were examined. Lipid peroxidation was quantified as a marker of oxidative stress using the OxiSelect 4-HNE Adduct Competitive ELISA Kit (Cell Biolabs, Inc., San Diego, CA, USA).

Cells were lysed with SDS sample buffer [100 mM Tris-HCl (pH 6.8) (Sigma Aldrich), 4 per cent SDS (Nippon gene, Tokyo, Japan), 20 per cent glycerol (Nacalai tesque), 10 per cent 2-mercaptoethanol (Wako)]. The cell lysates were subjected to SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Merck Millipore; Darmstadt, Germany). After blocking the membranes with 5 per cent skim milk (Morinaga, Tokyo, Japan), they were reacted with anti-tgDeV DAg, anti-mmDeV DAg, or anti-actin (Sigma Aldrich) antibodies as primary antibodies, followed by reaction with horseradish peroxidase (HRP)–conjugated secondary antibodies (Cell Signaling Technology, Danvers, MA).

Nourseothricin was purchased from Jena Bioscience (Dortmund, Germany). Cefotaxime sodium, D-glucose, agar, NaCl, KCl, sorbitol, H₂O₂, DTT, MMS, and AMPH-B were purchased from Fujifilm Wako Pure Chemical Industries (Osaka, Japan). Hygromycin B, caffeine, FLCZ, VRCZ, and N-phenylthiourea were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Congo Red and menadione were purchased from Sigma-Aldrich (St. Louis, MO, USA). G418 was purchased from Enzo Life Science, Inc. (Farmingdale, NY, USA). Hipolypeptone was purchased from Nihon Pharmaceutical Co., Ltd. (Tokyo, Japan). Tunicamycin (TM) was purchased from Cayman Chemical Company (Ann Arbor, MI, USA). Sodium dodecyl sulfate (SDS) was purchased from Nippon Gene Co., Ltd. (Tokyo, Japan). Tween 80 was purchased from MP Biomedicals LLC (Santa Ana, MO, USA).