TCR-zeta chain phosphorylation.
Purified naïve, adaptively tolerant, or preactivated T cells were stimulated for 1 min with varying concentrations of anti-TCRβ-biotin mAb (0, 1, or 10μg/ml) and anti-CD4-biotin mAb at either 0 or 10 μg/ml, followed by cross-linking with streptavidin. Samples were lysed with 1% NP40 buffer, immunoprecipitated with anti-ζ, mAb and run on SDS-PAGE. Western blots were performed and probed with anti-phosphotyrosine mAb and then reprobed with anti-Zap70 mAb or anti-ζ mAb.