Downstream signaling is altered in Syk-expressing p116 cells. (A) Erk phosphorylation in the parental p116 cells and the derived ZAP^E/Syk^– and ZAP^–/Syk^E cell lines was monitored 3, 5, and 10 minutes after stimulation with an αCD3 mAb at 37° C. Cell lysates (1 × 106 cell equivalents) were immunoblotted with a polyclonal Ab that recognizes the doubly phosphorylated forms of Erk1 and Erk2. The blot was then stripped and reprobed with an αErk2 mAb. (B) CD69 expression was assessed following overnight culture in serum-free media in the absence or presence of immobilized αCD3 mAb (1 μg/mL). Constitutive (filled histograms) and CD3-induced (open histograms) CD69 expression in the parental p116 cells and the derived ZAP^E/Syk^– and ZAP^–/Syk^E cell lines were detected using a phycoerythrin (PE)–conjugated anti-CD69 mAb and analyzed on a FACScan. The mean fluorescence intensity of CD69 expression in the absence or presence of αCD3 is indicated in each histogram.