Mutant SOD::YFP proteins form visible inclusions in cell culture. A) HEK293FT cells, on poly-L-lysine coated glass coverslips, were transfected for 24 hours as described in Methods. Cells were fixed and observed under a fluorescence microscope. All pictures were taken using a 40x objective, scale bars = 50 μm. The images shown are representative of at least 3 independent transfections for each construct. B) Immunoblot of P2 and S1 protein fractions of cells expressing SOD1::YFP proteins for 24 hours. The SOD1::YFP protein migrates at a size of approximately 50 kDa (filled arrowhead), while endogenous WT SOD1 monomer runs at 16 kDa (open arrowhead). C) Quantification of the aggregation propensity (P2/S1) of cells expressing SOD1::YFP proteins (see Methods). As expected, cells transfected with WT::YFP or mutant SOD1::YFP accumulated much more insoluble protein than untransfected cells: *p ≤ 0.05; #p ≤ 0.005. The aggregation propensity of A4V::YFP and G37R::YFP were both statistically greater than that of WT::YFP (#p ≤ 0.005). The data were averaged from at least 3 independent repetitions of each transfection and immunoblot.