Generation of T-cell lines expressing equivalent levels of ZAP-70 and Syk. (A) p116 Jurkat cells, expressing neither ZAP-70 nor Syk (ZAP^–/Syk^–), were transduced with a ZAP-70/EGFP or Syk/EGFP retroviral vector, and cells expressing similar levels of EGFP were sorted on a FACS Vantage cytometer. The EGFP fluorescence of the sorted p116 cell lines expressing ectopic ZAP-70 (ZAP^E/Syk^–) and Syk (ZAP^–/Syk^E) are shown. Background fluorescence of p116 cells not expressing EGFP is shown in a filled histogram. (B) ZAP-70 and Syk expression in these cells was quantified by comparison with known concentrations of human recombinant ZAP-70 and GST-Syk proteins (2-50 ng). Amido-black staining of these recombinant proteins and the corresponding immunoblots with Syk and ZAP-70 antibodies are shown. The quantification of ectopic ZAP-70 and Syk expressed in the transduced cell lines (A) was determined by comparison of the respective signals in several cell concentrations (15-60 × 103 cells) with that obtained for the recombinant protein (using the NIHimage software program). (C) ZAP-70 and Syk expression in the ZAP^E/Syk^– and ZAP^–/Syk^E cells as well as in other Jurkat cell lines (Table 1) were monitored by immunoblotting of total cellular lysates with αZAP-70 and αSyk Abs. The blot was then stripped and reprobed with an αErk2 mAb to control for protein loading.