Figure 3.

A, TCR competition assay. TCR-deficient Jurkat RT3-T3.5 cells were electroporated with 1 μg of each chain of the MART-HH (white columns) or MART-HM (gray columns) along with 1 μg of each chain of the competitor TCR [for p53-MH, we also used an additional amount (i.e., 0.2 μg) indicated as p53-MH (0.2)]. Twenty-four hours after electroporation, the cells were stained with MART-1 tetramer and the percentage of MART-1 TCR relative expression was calculated by dividing percentage of tetramer-positive cells of a given sample by that of the control sample (without competitor TCR). All the differences were statistically significant based on Student’s t test (P < 0.005). B, enhanced CD3/TCR stability mediated by MART-HM. TCR-deficient Jurkat RT3-T3.5 cells were electroporated with the MART-HH or MART-HM. Twenty-four hours after electroporation, cells were lysed in two different detergents (Brij96, mild detergent; NP40, strong detergent). The TCR was immunoprecipitated with a Vβ12 and the precipitate was subjected to a Western blot analysis for CD3ζ. As a control for protein loading (CD3ζ), we used the unprecipitated cell lysate. Representative of one of three independent experiments. C, enhanced CD3/TCR stability mediated by p53-MM. Similarly, we electroporated Jurkat RT3-T3.5 cells with either p53-MM or p53-MH and immunoprecipitated the TCR with an anti-murine Vβ3 antibody under different detergent conditions. We then subjected the precipitated to a Western blot analysis for CD3ζ and used the unprecipitated lysate as control.