Figure 3

Activation of the transgenic 9xNFAT-Luc reporter in fresh and mitogen-activated lymphocytes. A) Proliferating transgenic 9xNFAT-Luc T cells were exposed to increasingly hypertonic media in the presence of FK506, or stimulated with PMA plus ionomycin during 24 hours. Left panel: luciferase activity (RLU) shown is the mean ± S.E.M of five independent experiments. Right panel: NFAT5 and pyruvate kinase (protein loading control) were detected by Western blotting. One representative experiment is shown out of three performed independently. B) Luciferase activity in 9xNFAT-Luc transgenic thymocytes exposed to increasingly hypertonic media in the presence of FK506, or stimulated with PMA plus ionomycin during 24 hours. Mean ± S.E.M of three independent experiments is shown. C) Luciferase activity in 9xNFAT-Luc transgenic splenocytes stimulated during 24 hours with hypertonicity or PMA plus ionomycin, either immediately after their purification, or after a 24-hour preactivation with concanavalin A plus IL2. One representative experiment is shown out of three performed independently.