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Fig. 2. Activation of NFAT3 by TNF- in Cl41 cells. (A) 8x10³ Cl 41-NFAT Luc cells were seeded into each well of a 96-well plate. After being cultured at 37°C overnight, cells were treated with 5 or 20 U/ml of TNF- in combination with or without 10 µM CsA for 24 hours. The luciferase activity was measured as indicated in Fig. 1A. The results were expressed as the relative NFAT activity. Each bar indicates the mean and standard deviation of triplicate wells. (B) Cl41 cells (2x10⁵) were seeded into each well of a 6-well plate, and cultured in 5% FBS MEM at 37°C. When the cell density reached 70-80%, the cells were exposed to various concentrations of TNF- for 24 hours, and then washed once with ice-cold PBS and extracted with SDS-sample buffer. The NFAT3 protein levels in the extracts were detected by western blot. (C) Cl41 cells were treated with 20 U/ml TNF- for 12 hours, and then extracted, as described in Materials and Methods. The NFAT3 protein levels in cytoplasm (C) and nuclear extracts (N) were detected by western blot.

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