Quantification of total ZAP-70 and phosphorylated ZAP-70 after antigen–(in)dependent stimulation of TCR gp100-transduced T cells. (A) Hu T cells, transduced with a panel of indicated TCR gp100(280-288)–specific constructs, were stimulated with anti-Mu TCRβ or gp100(280-288)A2.1 tetramers in 6 independent experiments and cell extracts were submitted to enzyme-linked immunosorbent assay (ELISA) to quantify total ZAP-70. (B) TCR gp100-transduced T cells were treated with either a nonstimulating Mu Vβ3-specific antibody as isotype control or specifically stimulated with anti-Mu TCRβ by Cβ-domain cross-linking and analyzed for their amounts of total ZAP-70. For this, the mean OD₄₅₀ changes for each unspecifically, that is, anti-Mu Vβ3, stimulated, and TCR gp100-transduced T-cell population were set to one. The relative changes in absorbance on specific () versus unspecific stimulation () are indicated as stacked bars. Results were from 2 independent experiments. (C) Hu T cells transduced with the enumerated TCR gp100(280-288)–specific constructs were specifically stimulated with anti-Mu TCRβ in 6 independent experiments. In addition, murinized dcTCR gp100 was treated with irrelevant Mu Vβ3-specific antibody. Amounts of phosphorylated Y319 in ZAP-70 were quantified by ELISA. (D) TCR gp100-transduced Hu T cells were antigen-specifically stimulated with gp100(280-288)A2.1 tetramers or Hu Chim TCR gp100 was treated with an irrelevant p53(264-272)A2.1 tetramer. The indicated amounts of phosphorylated Y319 of ZAP-70 were the mean of 4 independent experiments. (A-D) The measured absorbances were converted to relative OD₄₅₀ changes normalized to the sample with the highest absorbance in each experiment to merge replicate experiments conducted on subsequent days. Error bars represent SD.