SOD1 activity assays.

For the in vitro colorimetric activity assay, HEK293T cells were either transfected with a FLAG-SOD1 plasmid or mock transfected. Cells were harvested and lysed, and FLAG was immunoprecipitated with FLAG epitope-conjugated agarose beads, after which the resin was washed three times in cold PBS. FLAG-SOD1 was then competitively eluted from the beads by incubation in a 200-ng/μl solution of purified FLAG peptide (ApexBio) with gentle shaking for 10 min at 4°C. Beads were then centrifuged at 8,200 × g for 30 s at 4°C. The FLAG-SOD1-containing supernatant was transferred to a new microcentrifuge tube; the elution was repeated; and the supernatant was added to the same tube. With the purified SOD1 or mock supernatants, SOD1 ROS scavenging activity was indirectly measured using a SOD assay kit purchased from Sigma (catalog no. 19160) according to the manufacturer's recommendations.

For the in situ SOD1 activity gel, purified FLAG-SOD1 was produced as in the colorimetric assay. SOD1 ROS scavenging activity was measured indirectly as a competitive reaction of superoxide with nitroblue tetrazolium (NBT) dye as described previously (65). Briefly, a portion of the purified SOD1 supernatants was mixed with 5× SOD loading dye (25 mM Tris-HCl [pH 6.8], 50% glycerol, 0.5% bromophenol blue) to a final concentration of 1× loading dye. Samples were then loaded onto an 8% native PAGE gel, and SOD1 was resolved at a constant 80 V until the loading dye reached the end of the gel. The gel apparatus was maintained at 4°C. The gel was then moved to a shallow container, immersed in 50 ml of staining solution (45 mM K₂HPO₄, 4.6 mM KH₂PO₄, 0.163 mM NBT, 0.266 mM riboflavin), and covered with foil. A 50-μl volume of N,N,N′,N′-tetramethylethylenediamine (TEMED) was then added, and the gel was incubated with gentle shaking for 1 h in the dark. Following incubation, the staining solution was removed, and the gel was first washed twice with distilled water and then immersed and incubated in distilled water with gentle shaking overnight, with exposure to ambient light. Clear bands on the blue-stained gel were interpreted as active SOD1. NBT (catalog no. N6639), K₂HPO₄ (catalog no. P3786), KH₂PO₄ (catalog no. P0662), and riboflavin (catalog no. R9504) were purchased from Sigma.