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. 1998 Aug 3;188(3):527–537. doi: 10.1084/jem.188.3.527

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(A and B) Effect of V12Rac-1 upon NFATC1-GFP nuclear accumulation in RBL2H3 mast cells. RBL2H3 cells were transfected with 8 μg NFATC1-GFP reporter alone or in combination with 20 μg pEF-V12Rac-1. Cells were recovered for 6 h on glass coverslips before IgE priming and stimulation for 30 min with the indicated concentrations of KLH-DNP (A) or stimulation with 500 ng/ml KLH-DNP for the indicated times (B). Cells were fixed, mounted, and scored for predominant localization of GFP as described. (C) Effect of V12Rac-1 on NFATC1-GFP nuclear export in RBL2H3 mast cells. RBL2H3 cells were transfected with 8 μg NFATC1-GFP reporter alone or in combination with 20 μg pEF-V12Rac-1. Cells were recovered for 6 h on glass coverslips before stimulation for 30 min with 500 ng/ml Ionomycin to induce nuclear uptake of NFATC1-GFP. The medium was then exchanged twice with warm DMEM/10% FCS before addition of 50 nM CsA to prevent further import. At the indicated time points after CsA addition, cells were fixed, mounted, and scored for predominant localization of GFP as described.

(A and B) Effect of V12Rac-1 upon NFATC1-GFP nuclear accumulation in RBL2H3 mast cells. RBL2H3 cells were transfected with 8 μg NFATC1-GFP reporter alone or in combination with 20 μg pEF-V12Rac-1. Cells were recovered for 6 h on glass coverslips before IgE priming and stimulation for 30 min with the indicated concentrations of KLH-DNP (A) or stimulation with 500 ng/ml KLH-DNP for the indicated times (B). Cells were fixed, mounted, and scored for predominant localization of GFP as described. (C) Effect of V12Rac-1 on NFATC1-GFP nuclear export in RBL2H3 mast cells. RBL2H3 cells were transfected with 8 μg NFATC1-GFP reporter alone or in combination with 20 μg pEF-V12Rac-1. Cells were recovered for 6 h on glass coverslips before stimulation for 30 min with 500 ng/ml Ionomycin to induce nuclear uptake of NFATC1-GFP. The medium was then exchanged twice with warm DMEM/10% FCS before addition of 50 nM CsA to prevent further import. At the indicated time points after CsA addition, cells were fixed, mounted, and scored for predominant localization of GFP as described.