Dark blue cathode buffer

Crude mitochondrial fractions were isolated and normalized for total protein content as described above. Blue Native (BN) PAGE was performed using the Invitrogen NativePAGE system. 100 µg of mitochondria were pelleted at 10,000 x g for 10 min at 4°C and resuspended in 1x pink lysis buffer (Invitrogen, BN20032). Digitonin (GoldBio D-180–2.5) was added to a final concentration of 1% mass/volume. Samples were incubated on ice for 15 min, then spun for 20 min at 20,000 x g. 6 µL of NativePAGE sample buffer (Invitrogen, BN20041) was added and 10 µL of sample was run on precast 3–12% NativePAGE gels (Invitrogen, BN2011B × 10) with NativePAGE anode buffer (Invitrogen, BN2001) and dark blue cathode buffer (Invitrogen, BN2002) at 150 V for 1 hr then switched to light blue cathode buffer (Invitrogen, BN2002) and run at 30 V overnight. Gels were subsequently transferred to PVDF at 100 V, washed with methanol, and blotted with the indicated primary antibodies which are listed in the key resources table according to the manufacturers’ recommendations. Secondary anti-mouse HRP antibody listed in the key resources table and SuperSignal West Femto Maximum Sensitivity Substrate (Thermo, 34096) was used to visualize bands on film (GeneMate, F-9024−8 × 10).

Purified human α₂-macroglobulin protein (725 kDa) was obtained from Enzo Life Science (New York, USA), anti-human monoclonal mouse α₂-macroglobulin (immunoglobulin (Ig)G1 clone) from R&D Systems (Minneapolis, MN, USA), and goat anti-mouse IgG (H + L)-horseradish peroxidase (HRP) conjugate from Bio-Rad Laboratories (Hercules, CA, USA). Native PAGE™ Sample Buffer, Native PAGE Running Buffer, Dark Blue Cathode Buffer, Native Mark^™ Unstained Protein Standard, and Light Blue Cathode Buffer were obtained from Thermo Fisher Scientific (Waltham, MA, USA). DTT, Blocking One and Peroxidase Stain 3,3′-Diaminobenzidine kit (Brown Stain) were purchased from Nacalai Tesque (Kyoto, Japan). Immune-Blot^® polyvinylidene difluoride membrane, Sequi-Blot™ membrane, Precision Plus Protein™ Dual Color Standards and Mini-PROTEAN^® TGX™ were obtained from Bio-Rad Laboratories. Perfect NT Gel, Perfect NT Gel System and SDS-PAGE Running Buffer were obtained from DRC (Tokyo, Japan), and ECL Prime Western Blotting Detection Reagent from GE Healthcare (Buckinghamshire, UK).

G93A mice and their WT litters were injected daily with vehicle or compound 2a (10 mg/kg, from day 83 to day 103). Lumbar spinal cords dissected from saline-perfused mice were lysed in Tris-HCl 10 mM pH 8, EDTA 1 mM pH 8, NaCl 100 mM, NP40 1% and protease inhibitor cocktail (Sigma–Aldrich). Extracts were obtained using a tight-fitting glass Potter tissue grinder (1 mL; Wheaton). We incubated 250 µL of each extract with 100 mM Iodoacetamide (Sigma–Aldrich). Samples were mixed with 4× Sample NativePAGE (Thermofisher) sample buffer and the G-250 additive (Thermofisher) and proteins were resolved on native 4–15% Bis-Tris gel (Bio-Rad) with Dark Blue Cathode buffer (Thermofisher) at 150 V for 1/3 of gel length. We replaced the running buffer with Light Blue Cathode Buffer to complete the run. At the end of the electrophoresis, gels were washed with 5 mM tris(2-carboxyethyl)phosphine (Sigma–Aldrich) for 15 min and then proteins were transferred to nitrocellulose and fixed on the membrane by 8% acetic acid for 15 min. Rabbit α-SOD1 (1:2000, Genetex) was incubated overnight as above described. Visualization of proteins was performed using ClarityMax-ECL (Bio-Rad) according to the manufacturer’s instructions and images were acquired on ChemiDoc (Bio-Rad).

Characterization of the protein assemblies in native conditions was performed on 4–16% Native-PAGE Bis-Tris gels (Thermo Fisher Scientific). Samples were mixed with 4x Native PAGE sample buffer (Thermo Fisher Scientific) and run in the Dark Blue Cathode Buffer (Thermo Fisher Scientific) for 60 min at 150 V and for 40–60 min at 250 V according to the manufacturer instructions. The gels were fixed with 40% methanol (v/v) and 10% acetic acid (v/v) and destained with 8% acetic acid (v/v).