Overexpression of wild-type SHP-1 negatively modulates the bpV-mediated signal transduction pathway leading to NFAT activation.

Jurkat cells were transiently transfected by the DEAE-Dextran protocol with 15 μg pNFAT-LUC and increasing quantities (0 μg, ■; 7.5 μg, ░; and 15 μg, ▪) of a vector encoding for the wild-type (A) or a dominant-negative version (B) of SHP-1. The total amount of DNA for each transfection was kept constant by the addition of filler DNA. Following a 24-hour incubation period, cells were left untreated or stimulated with either PHA (3 μg/mL), PHA (3 μg/mL)/PMA (20 ng/mL), or bpV[pic] (10 μM) for 8 hours. Cells were then lysed, and luciferase activity was monitored as described in “Materials and methods.” Results are the means ± SD for triplicate samples and are representative of 3 independent experiments. (C) Jurkat (lanes 1-4) or J.SHP-1C/S (lanes 5-8) cells were either left untreated (lanes 1-2 and 5-6) or stimulated with PMA (20 ng/mL)/Iono (1 μM) (lanes 3-4 and 7-8) for 1 hour. Nuclear extracts were then prepared as described in “Materials and methods,” and 10 μg was incubated with the γ³²P-labeled NFAT probe for 20 minutes in the absence (lane 1, 3, 5, and 7) or presence of 100-fold excess of unlabeled NFAT oligonucleotide (lanes 2, 4, 6, and 8). Samples were then migrated on a native polyacrylamide gel, dried, and exposed on Kodak X-OMAT film. Free probe and specific complexes are indicated on the left side of the panel.