Detergent-insoluble forms of mutant SOD1 remain cell associated after saponin treatment. A) Immunoblots of HEK293FT cells that were transfected with the indicated SOD1 constructs for 48 hours. Prior to harvest, cells were incubated 30 minutes in PBS with 0.01% saponin. The harvested cell pellets were analyzed by detergent extraction and centrifugation assays (S1 fractions, each lane contains 5 μg of total protein; P2 fractions, each lane contains 20 μg of total protein). The amount of SOD1 protein released into the cell media was also evaluated (each lane loaded with 5 μg of total protein). B) Quantification of SOD1 protein found in the Media, P2 and S1 fractions of cells untreated or treated with saponin (Sap.), and expressing WT or A4V. Symbols over bars indicate differences from corresponding non-treated control. Unpaired t-tests: *p ≤ 0.05. The data were averaged from at least 3 independent transfection and immunoblotting experiments.