Fig. 2

Detection of SOD1^SO3H in CSF of ALS cases using anti-SOD1^SO3H antibody. (a) Native SOD1 (Sigma, #S9636: 20 μU) and a recombinant sample containing SOD1^SO3H (2.5 ng) were analyzed by Western blotting side-by-side with (left) anti-SOD1^SO3H and (right) FL-154 antibodies. The recombinant sample containing SOD1^SO3H produced doublet bands in the Western blot with FL-154 (right), but only the upper band was recognized with anti-SOD1^SO3H 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-SOD1^SO3H antibody (left). These results confirm the retarded electrophoretic mobility of SOD1 upon sulfonylation of Cys and also support the specificity of our anti-SOD1^SO3H antibody toward SOD1^SO3H. (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-SOD1^SO3H 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-SOD1^SO3H antibody and also exhibited the same electrophoretic mobility with that of recombinant SOD1^SO3H (20 ng). (c) CSF samples (4 μg of total proteins) and recombinant SOD1^SO3H (10 ng) were analyzed by Western blotting with anti-SOD1^SO3H 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-SOD1^SO3H (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)