FIG. 5.

Effects of overexpressed cDNA on secretion levels of scTCRs and scFvs. (A) Representative Western blotting data for 4-4-20 scFv secretion experiments. Triplicate supernatants derived from independent transformants were used to generate the Western blotting signals. Within each temperature condition, the data shown are from the same time point of exposure and can therefore be directly compared. However, these only serve as qualitative comparisons, while quantitative values and associated statistical significance can be found in panels B and C (see Materials and Methods for quantitation details). WT, wild type. (B) Secretion yields after induction at 20°C. (C) Secretion yields after elevated temperature induction. As indicated in panel C, each cDNA overexpression strain was evaluated at the elevated induction temperature at which it was selected. 7/15 secretion levels were detected by quantitative ELISA, while all other protein levels were measured by quantitative Western blotting, as depicted for 4-4-20 in panel A. Activity assays confirmed that increased secretion of 4-4-20 and LWHI expression detected via Western blotting was representative of active protein levels. Single and double asterisks represent P < 0.05 and P < 0.1, respectively, and were reproducibly significant over three independent experiments. Triple asterisks indicate data that were statistically significant (P < 0.05) for the experiment presented, but were not reproducibly significant over multiple experiments. These *** conditions were therefore reported as incapable of increasing secretion levels. Triplicate independent transformants were evaluated for each protein-cDNA combination, and all data were normalized to the wild type at the corresponding temperature.