Analysis of detergent solubility and inclusion formation in cells co-expressing WT and mutant SOD1. HEK293FT were transfected with the indicated SOD1 constructs for 24 (A, B, E, G) or 48 (C, D, F, H) hours as noted above the figure. A, C) Immunoblots of P2 and S1 fractions of HEK293FT cells co-transfected with the indicated SOD1 constructs for 24 (A) or 48 (C) hours and immunoblotted with a SOD1 antibody. Tagged SOD1::YFP proteins migrate on SDS-PAGE at relative molecular mass of 50 kDa, while untagged SOD1 variants migrate at about 16 kDa. B, D) Quantification of the aggregation propensities (P2/S1) of tagged and untagged proteins from singly or doubly transfected cells at 24 (B) and 48 (D) hours. Statistical comparisons were calculated through unpaired student t-tests, *p ≤ 0.05, #p ≤ 0.005. The data were averaged from at least 3 independent transfection and immunoblotting experiments. E-H) Cells transfected for 24 (E, G) or 48 hours (F, H) were fixed and visualized under a confocal microscope. Scale bars = 20 μm. The images shown are representative of at least 3 independent transfection experiments.