FIG. 8.

Reconstitution of gene transcription in P116 cells expressing ZAP-70 or Syk. (A) Effect of Syk versus ZAP-70 on NFAT-driven gene transcription. P116 cells were transfected with 10 μg of pNFAT-Luc in the presence of empty vector (mock) or the indicated amounts of pcDNA3-mZAP or pcDNA3-mSyk. After transfection, equivalent samples of each cell population were stimulated for 6 h with medium only (unstim) or with 1 μg of anti-CD3 antibody (OKT3) per ml. Cellular extracts were assayed for luciferase activity and for expression of epitope-tagged proteins by immunoblotting with 9E10 MAb. The results shown are representative of four separate trials. (B) TCR-dependent effects of Syk and ZAP-70 on NFAT-driven gene transcription. P116 and TCR-negative J.RT3 cells were transfected with 10 μg of pNFAT-Luc and 10 μg of empty vector or the indicated plasmid. Samples were processed as in panel A. The results shown are representative of three independent experiments.