Figure 4.

Inhibitory TCR-ζ mutants are tyrosine phosphorylated. (a) hCD8-ζ mutants 4F and Wt are tyrosine phosphorylated before and after cross-linking of hCD8α. 2 × 10⁷ untransfected 3.L2 hybridoma cells or 3.L2 cells transfected with 6F, 4F, or Wt were incubated with 5 μg of anti-hCD8α OKT-8 on ice. Samples were then activated and cross-linked at 37°C (+) or left on ice (−). Cells were lysed, and the mutants were immunoprecipitated using OKT-8 and analyzed by phosphotyrosine blotting using biotin–4G10 (Upstate Biotechnology Inc.) and horseradish peroxidase–streptavidin. The position of a 35-kD molecular mass marker is indicated at left. (b) 3.L2 cells transfected with 6F, 4F, and Wt express hCD8-ζ protein. An immunoblot was performed on the samples described in panel a using 777, an antibody against murine ζ that also recognizes mutant ζ 11. Two bands with different molecular sizes were apparent, most likely due to glycosylation of the extracellular portion of hCD8α. Representative of four experiments.