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Fig. 2 | Molecular Neurodegeneration

Fig. 2

From: Wild-type Cu/Zn-superoxide dismutase is misfolded in cerebrospinal fluid of sporadic amyotrophic lateral sclerosis

Fig. 2

Detection of SOD1SO3H in CSF of ALS cases using anti-SOD1SO3H antibody. (a) Native SOD1 (Sigma, #S9636: 20 μU) and a recombinant sample containing SOD1SO3H (2.5 ng) were analyzed by Western blotting side-by-side with (left) anti-SOD1SO3H and (right) FL-154 antibodies. The recombinant sample containing SOD1SO3H produced doublet bands in the Western blot with FL-154 (right), but only the upper band was recognized with anti-SOD1SO3H antibody (left). Also, native SOD1 without any oxidation was detected as a single band in the Western blot with FL-154 (right) but was not recognized with anti-SOD1SO3H antibody (left). These results confirm the retarded electrophoretic mobility of SOD1 upon sulfonylation of Cys and also support the specificity of our anti-SOD1SO3H antibody toward SOD1SO3H. (b) The CSF sample from ALS7 (4 μg of total proteins; ALS7 concd. Sample contained 10 μg of total proteins) was analyzed by Western blotting side-by-side with (left) anti-SOD1SO3H and (right) FL-154 antibodies. Doublet SOD1-positive bands were observed in the Western blot probed with FL-154 (right), among which only the upper band was recognized with anti-SOD1SO3H antibody and also exhibited the same electrophoretic mobility with that of recombinant SOD1SO3H (20 ng). (c) CSF samples (4 μg of total proteins) and recombinant SOD1SO3H (10 ng) were analyzed by Western blotting with anti-SOD1SO3H antibodies. In all panels, the blots on the membranes were reacted with either FL-154 (0.2 μg/mL) in PBS-T with 1% (w/v) skim milk or anti-SOD1SO3H (0.06 μg/mL) in PBS-T with 3% (w/v) BSA, and then probed with anti-rabbit secondary antibody in PBS-T with 0.5% (w/v) skim milk (1:2000 dilution)

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