Chemidoc touch apparatus

Western blots were performed on Any kD or 18% (w/v) Criterion TGX precast gels (BioRad Laboratories, Hercules, CA, USA) as previously described [51 (link)]. Primary antibodies (see below) were incubated overnight at 4 °C. Horseradish peroxidase (HRP)-conjugated secondary antibodies (anti-mouse or anti-rabbit IgG; 1:10,000, Dako, Glostrup, Denmark) were incubated for 1 h at room temperature. ECL Select reagent (GE Healthcare Biosciences, Piscataway, NJ, USA) was used to detect the signal, which was recorded on a ChemiDoc Touch apparatus (BioRad Laboratories, Hercules, CA, USA) and analyzed using ImageLab software (BioRad Laboratories, Hercules, CA, USA).
The primary antibodies used for western blotting were; rabbit anti-human SOD1 antibodies raised against peptides corresponding to aa 24–39 (2.3 μg/ml), aa 57–72 (1.6 μg/ml), aa 144–153 (5.2 μg/ml) and aa 123–127 GQRWK (4.8 µg/ml, G127X-specific) [46 (link)], rabbit anti-CCS raised against peptides corresponding to aa 252–270 of the human CCS sequence (CCS, 0.9 μg/ml) [46 (link)], rabbit anti-CCS (1:1000 Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-glutaredoxin-1 (1:250, Abcam, Cambridge, UK), mouse anti-β-actin (1:200,000; Millipore, Bedford, MA, USA) and rabbit anti-GAPDH (1:1000, Abcam, Cambridge, UK).

Western blot analysis was performed on muscle homogenates as previously described [16 (link)]. Briefly, after electrophoretic separation on a 4–20% gradient acrylamide gel (Stain-free precast gel, Biorad, France) and electrotransfer to Immobilon P (Biorad, France), the membrane was incubated with primary antibodies and then HRP-labelled secondary antibodies (Jackson ImmunoResearch Laboratories). Signal quantification was performed using a ChemiDoc Touch apparatus (Biorad, France) and the Image Lab software (Biorad). The amount of the chosen protein in each sample was corrected for differences in loading using either the amount of myosin, GAPDH or the total amount of proteins using the stain free system from Biorad, and normalized to the amount of the same protein present in the control, set to 100% as described previously [15 (link)]. Ten human controls (muscle biopsy from individuals non-affected by neuromuscular disease) of different age have been used, from 3.5 to 64 years. For each sample (mouse and human), 2–3 Western blots have been performed, and the value for each sample corresponds to the mean ± SEM of the different Western blots.

Muscle (quadriceps) or brain homogenates were prepared as described previously from frozen tissues [14 (link)]. The presence and the amount of different proteins in tissue homogenates was assayed by Western blot analysis, using a chemiluminescent reagent after electrophoretic separation of the protein on a 5–15% acrylamide gel, and electrotransfer on Immobilon P (Millipore) [39 (link)]. The quantification was performed using a ChemiDoc Touch apparatus (Biorad, Marnes-la-Coquette, France) and the ImageLab Software (Biorad). The signal was normalized to the amount of a muscle-specific protein of low abundance (triadin T95) or GAPDH.