Fig. 6

Misfolded SOD1 specific antibodies show preferential reactivity for demetallated (apo) SOD1. a Recombinant SOD1 proteins (WT, G93A, G85R and A4V) were produced (i) with its full complement of metals (holo); (ii) with its full complement of metals and incubated so as to produce low levels of aggregated protein (holo inc); (iii) lacking metals (apo); and (iv) lacking metals and a reduced Cys57-Cys146 disulfide bond (apo reduced) were spotted onto nitrocellulose and probed for misfolded SOD1 with AMF7-63 (left) and B8H10 (right). b In vitro mitochondrial binding assay. Recombinant SOD1^WT and SOD1^G93A were incubated with spinal cord mitochondria from a non-transgenic rat, washed and subjected to analysis by western blot. Recombinant SOD1 was either left untreated or incubated with EDTA or H₂O₂ before addition to mitochondria. c Quantification of b normalized to SOD1^G93A binding SOD1^WT (white) and SOD1^G93A (black). * P < 0.05, ** P < 0.01, n = 5