Defective basal and CD3-induced TCR-ζ chain phosphorylation in primary Syk⁺ T cells from a ZAP-70–deficient patient are restored following introduction of ZAP-70. CD4⁺ T cells from a healthy individual (ZAP⁺/Syk^–), a ZAP-70–deficient patient (ZAP^–/Syk⁺), and following introduction of ectopic ZAP-70 (ZAP^E/Syk⁺)^27,45  were either left unstimulated (–) or stimulated for 3′ with a cross-linked αCD3 mAb (+). (A) Phosphorylation of the TCR-ζ chain was monitored in whole cell lysates by the presence of the p21 and p23 TCR-ζ isoforms. Nonphosphorylated TCR-ζ migrates with a molecular weight of 16 kDa (p16). ZAP-70 and Syk levels in these whole cell lysates were assessed with ZAP-70– and Syk-specific Abs, and the relative amount of protein in each lane was determined by blotting for Erk2. Note that the ectopic ZAP-70 bears a terminal vesicular stomatitis virus-protein G epitope tag allowing endogenous and ectopic ZAP-70 proteins to be distinguished on the basis of their molecular weights. (B) TCR-ζ was immunoprecipitated from unstimulated (–) or αCD3/αCD4-stimulated (3′, +) control (ZAP⁺/Syk^–), and patient (ZAP^–/Syk⁺) CD4⁺ T cells. The levels of ζ-associated ZAP-70 and Syk molecules were determined by immunoblotting with αZAP-70 and αSyk Abs, and tyrosine phosphorylation of these proteins was monitored by immunoblotting with an α-phospho-tyrosine (PY) mAb. The amount of immunoprecipitated p16-ζ in each lane was monitored with an α-ζ mAb.