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).
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).
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