Analysis of C4F6 binding by SOD1 variants encoding an experimental K91E mutation in G93A hSOD1.

CHO cells were transfected with plasmids for each SOD1:YFP variant and after 24 or 48 hours the cells were fixed and immunostained as described in Methods. One image of direct YFP fluorescence was captured before a second image of C4F6 immunoreactivity (red) was captured, using a conventional epifluoresence microscope (20x magnification). Cells transfected with WT-SOD1:YFP serve as a negative control and cells transfected with G93A-SOD1:YFP serve as a positive control (see Fig 4 for an example of 48 hours post-transfection). Each transfection was repeated at least 3 times. For the 48 hour time point, we captured images from at least 3 fields of view and counted all cells expressing the YFP fusion protein and cells that were also labeled with C4F6. Mutation of Lys 91 to Glu in G93A-SOD1:YFP did not markedly diminish the frequency of cells binding the C4F6 antibody at 48 hours post-transfection.