Fig. 5. Verification of cells overexpressing SodA. (A) Exponentially growing parental Ax2 (A) and SOD-OE (B) cells (grown in 20 µg/ml G418) were lysed in SDS sample buffer and resolved by 12% SDS-PAGE, along with cell lysate from human Jurkat T cells (C) as a positive control, before being transferred to a PVDF membrane. The membrane was probed with polyclonal antisera raised against SOD (FL-154). A band of around 20 kDa, the expected size for SOD, was detected in both Ax2 and Jurkat cells. Increased immunoreactivity was seen for a doublet in the SOD-OE cells. The blot was then stripped and reprobed with antibody specific for the human c-myc epitope which had been inserted at the N terminus of the exogenous SodA protein, using the 9E10 antibody. This reacted with a single band, only in the SOD-OE cells, co-migrating with the upper of the two bands from the anti-SOD blot. It is likely that the lower band of the doublet seen with the anti-SOD antibody (co-migrating with the band seen in Ax2 cells) is SOD protein in which the N-terminal myc tag has been cleaved. The blot was then reprobed using the C-11 anti-actin antibody as a loading control. (B) 24-hour XTT assays were performed on the following cells aggregating on plastic as described in Fig. 1. GAL indicates Ax2 cells expressing ß-galactosidase, after selection at 100 µg/ml G418; 20, 50, 100 indicate Ax2 cells overexpressing SodA, (SOD-OE) after selection at 20, 50 or 100 µg/ml G418, respectively. Data are the means ± s.e.m. of three experiments.