Syk, but not the introduced ZAP-70, is phosphorylated in response to FcγRI cross-linking. (a) Uninfected U937 cells or U937 cells infected with the LN (U937/LN) or ZAP-70 (U937/ZAP-70) containing retrovirus were induced with IFN for 4 days. Cells were then either incubated alone (−) or prebound with the FcγRI Fab ′₂ MoAb (+) and cross-linked with rabbit α-mouse antibody. Lysates from 5 × 10⁶ cells were immunoprecipitated with a rabbit polyclonal anti-Syk antibody, fractionated on polyacrylamide gels, analyzed by immunoblotting with the 4G10 monoclonal antiphosphotyrosine antibody, and developed with ECL to assess the phosphorylation status of Syk. (b) Blots were then stripped and reprobed with a polyclonal rabbit anti-Syk antibody. (c and d) Cell lysates were similarly immunoprecipitated with a ZAP-70–specific rabbit polyclonal antibody and probed with an antiphosphotyrosine (c) or monoclonal ZAP-70 antibody (d). For comparison, the level of expression and tyrosine phosphorylation state of ZAP-70 was assessed in Jurkat T cells that were either unstimulated (−) or stimulated with a CD3-specific antibody (+). These data are representative of results obtained in six independent experiments.