FIGURE 5. Wild-type hSOD1 increases both mutant hSOD1 solubility and toxicity; thus increased toxicity can be rescued by upregulating Hsp70.

A) CHO cells were transiently transfected with equal total amounts of mutant hSOD1 (mut-mut) or mutant and wild-type-3xFLAG (mut-WtFLAG) hSOD1 DNA; 48 hours later protein was separated into readily soluble and detergent-soluble fractions, and detected on immunoblot with a pan-SOD1 antibody. Lane (1) = readily soluble mut-mut, (2) = detergent-soluble mut-mut, (3) = readily soluble mut-WtFLAG, (4) = detergent-soluble mut-WtFLAG; the Wt-3xFLAG protein has a slightly higher molecular weight. B) Bands were quantified using optical densitometry, confirming that co-expression with WT hSOD1 increases the proportion of readily soluble mutant protein for A4V and G93A. C) The increased proportion of readily soluble mutant SOD1 protein corresponds with toxicity. Wild-type hSOD1 induced toxicity in A4V and G93A expressing cells relative to the mut-mut condition. D) Induction of heat shock proteins with geldanamycin (10 μM for 24 hours) protects against toxicity created by co-expressing A4V with WT hSOD1 protein. For B and C, statistical comparisons are mut-WtFLAG vs. the mut-mut condition. For D statistical comparisons are with the DMSO condition for each hSOD1 mutant. Values represent the means of at least 3 independent experiments ± SEM (* = p<0.05, ** = p< 0.001, *** = p<0.0001).