Reconstitution of the ZAP70/Syk-deficient P116 cell line with wild-type ZAP70 restores both CD3- and CD2-mediated induction of NFAT transcriptional activity.
Wild-type Jurkat cells, the ZAP70/Syk-deficient clone P116 and the ZAP70-reconstituted P116 cells were subjected to transient transfection for evaluation of NFAT transcriptional activity by luciferase assay, as described in Figure 12. Twenty-four hours after transfection, 106 cells/test were then either left unstimulated (ø) or stimulated with plate-bound OKT3, T112+T113, or PMA plus ionomycin for 6 hours. Cells were then washed and lysed, and the soluble extract was assayed for luciferase activity. Results are reported as activity of firefly luciferase normalized to that ofRenilla luciferase (to correct for transfection efficiency) in the lysate, and expressed as percentage of response obtained with PMA plus ionomycin (P+I).
