Additional file 10: Figure S9. of Immunochemical characterization on pathological oligomers of mutant Cu/Zn-superoxide dismutase in amyotrophic lateral sclerosis

Anti-SOD1int antibody specifically detects pathological SOD1 in spinal cords of loxG37R mice. (A, B) SOD1 species recognized by (A) anti-SOD1int and (B) anti-SOD1 (FL-154, Santa Cruz Biotechnology) antibody were quantified in the soluble fraction of the homogenates of lumbar spinal cord (red), cervical spinal cord (green), brainstem (blue), and cerebellum (gray) of loxG37R mice by sandwich ELISA. Ages of the mouse samples examined are as follows; 100 (three independent mice), 174 (two independent mice), 188, 374 (three independent mice), 386, 392, and 445 days of age. The data were divided into two groups before and after the onset of the disease (350 days of age) and statistically analyzed by two-tailed student’s t test. The difference in the signal intensity before and after the disease onset was statistically significant in lumbar and cervical spinal cords (**: P < 0.01). (C) Soluble disulfide-crosslinked SOD1 oligomers in loxG37R mice were examined by Western blotting. Lumbar spinal cords of loxG37R and G1H mice at indicated days of ages were homogenized in the presence of 100 mM iodoacetamide and 1% NP-40 and centrifuged at 20,000 x g for 30 min so as to prepare soluble supernatant. In the presence and absence of the reducing reagent, β-ME, the supernatant was then separated in a polyacrylamide gel by SDS-PAGE and probed by Western blot using anti-SOD1 antibody (FL-154, Santa Cruz Biotechnology). GAPDH was used as a protein loading control for Western blot. (PDF 870 kb)