Biochemical changes after chimeric receptor stimulation.

Crosslinking induced chimeric receptor phosphorylation (A), lck association (B), and ZAP-70 phosphorylation (C) are shown. A quantity of 10⁷ transduced 4G4 cells were incubated with H-2K^b–specific antibody followed by crosslinking with goat antimouse IgG. Lysate was immunoprecipitated with a K^b, or ZAP-70–specific monoclonal antibody. Western blots were probed with antiphosphotyrosine (A, C) or anti-lck (B), stripped, and then probed with ζ-specific (A,B) or ZAP-70–specific (C) antibody to control for gel loading and membrane transfer. Representative analyses are shown. Arrowheads in (A) and (C) indicate tyrosine phosphorylated chimeric receptors. The position of phosphorylated ZAP-70 is shown in (C). As noted in the text, staining of the K^b-ζ-lck and K^b-CD28-ζ–lck receptors with ζ-specfic antibody was consistently poor, and likely reflects a diminished affinity of antibody for these chimeric receptors. In (A), the faint diffuse band present in the unstimulated lane of the phosphotyrosine analysis of K^b-CD4-ζ and migrating slightly lower than the K^b-CD4-ζ construct was not observed in 2 additional experiments and therefore likely represents crossreactive material.