Figure 1

The D90A ALS mutant binds C4F6 weakly. Transiently-transfected cells expressing hSOD1 proteins G93A (a, d), WT (b, e), or D90A (c, f) were immunostained with either hSOD antibody or C4F6 antibody. Nuclei were stained with DAPI (blue). (g) For immunoblot analysis, HEK293FT cells were transiently transfected with G93A, WT, D90A, or left untransfected (UT) for 48 hours and 50, 10, and 2 μg of total protein from homogenates were analyzed by SDS-PAGE. (h) Immunoblots similar to those shown in (g) were quantified. At each protein concentration, the most intense band (asterisk) was quantified and the intensity was normalized to the intensity for the band produced in cells expressing the G93A variant. The positions of endogenous SOD1 from CHO cells is marked as is the position of an unknown cross-reactive band. In cells transfected with constructs for G93A SOD1, a second faster migrating band was detected by both hSOD1 and C4F6 antibody (double asterisk). This band may be a cleavage product of G93A SOD1 or represent protein modified in some manner. Data from 3 replicate experiments were quantified and graphed (mean ratio ± S.E. (error bars)). Scale bar 50 μm. * P ≤ 0.05, *** P ≤ 0.001 (unpaired t-test).