Tyr319 and Tyr493 in ZAP-70 are inefficiently phosphorylated following IgM ligation in stable BJAB transfectants. (A) Immunoblotting analysis of immunoprecipitated tyrosine-phosphorylated proteins from parental and ZAP-70 transfectant BJAB B cells (unstimulated or anti-IgM stimulated) and Jurkat T cells (unstimulated or anti-CD3 stimulated). Membranes were probed with anti–ZAP-70 (top panel) and anti-Syk (bottom panel). The band corresponding to ZAP-70 in BJAB is 1 kDa bigger than the band corresponding to endogenous ZAP-70 in Jurkat T cells due to the presence of the Myc tag. The expected positions of ZAP-70, ZAP-70-Myc, Syk, and SykB are indicated by arrows. (B) ZAP-70 and Syk were immunoprecipitated from parental and ZAP-70–transfected BJAB B cells and from Jurkat T cells and analyzed by immunoblotting with phospho-ZAP-70^Tyr319/Syk^Tyr352 (top panels) or phospho-ZAP-70^Tyr493/Syk^Tyr526 antibody (bottom panels). The ratio of ZAP-70 phosphorylated at Tyr319 and total ZAP-70 was quantified by densitometry.