Relative binding of Syk to fully phosphorylated TCR-ζ is significantly lower than that of ZAP-70. (A) Induction of the phosphorylated p21 and p23 TCR-ζ isoforms was assessed in Jurkat cells expressing endogenous ZAP-70 and Syk (ZAP⁺/Syk⁺) following a 3′ CD3 ligation. Phosphorylation was monitored using an αPY mAb, and total levels of p16-ζ were assessed with an α-ζ mAb. (B) TCR-ζ was immunoprecipitated from lysates of 10 × 106 of these ZAP⁺/Syk⁺ Jurkat cells following CD3 cross-linking or pervanadate treatment, and the presence of associated ZAP-70 and Syk was detected by immunoblotting. To determine the relative levels of association of these 2 kinases with TCR-ζ, the total cellular levels of ZAP-70 and Syk present in 5 × 106, 1 × 106, and 0.5 × 106 of these Jurkat cells was concomitantly monitored on the same blot. (C) Western blots were quantified using scanning densitometry, and the relative levels of TCR-ζ–associated ZAP-70 and Syk in unstimulated and stimulated cells from the experiment shown in panel B are shown.