Fig. 5. Mutant SOD1 proteins affect ADP but not Ca²⁺ accumulation into mitochondria.

(A) ADP or Ca²⁺ accumulation into isolated mitochondria was measured using a filter trap assay with radiolabeled ⁴⁵CaCl₂ or [³H]ADP. Mitochondria were isolated from fresh spinal cords and livers of non-transgenic rats. (B) ADP and (C) Ca²⁺ accumulation were measured before and after the addition of 3 μM (50 μg/ml) hSOD1^wt, hSOD1^G93A or hSOD1^G85R purified proteins. Student's t test was used and p < 0.001 (marked by three asterisks) and p < 0.01 (marked by two asterisks) were considered statistically significant. Values represent the means ± SEM of three independent experiments. (D) Purified hSOD1^wt, hSOD1^G93A or hSOD1^G85R were incubated with liver or spinal cord mitochondrial fractions purified from a non-transgenic rat for 20 min at 37°C. The samples were then washed 3 times and the mitochondrial pellet was subjected to immunoblot using an SOD1 antibody. (E) Purified hSOD1^wt, hSOD1^G93A or hSOD1^G85R was incubated for 20 min at 37°C with spinal cord mitochondria purified from non-transgenic rats. The samples were then washed 3 times and the mitochondrial pellet was subjected to immunoprecipitation using DSE2 (3H1) antibody, a monoclonal antibody only recognizing misfolded SOD1. The immunoprecipitates were immunoblotted using an SOD1 antibody.