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. 2018 Feb 18;19(2):605. doi: 10.3390/ijms19020605

Figure 2.

Figure 2

Evaluation of the NanoLuc reporter system by expressing murine NFAT1. (A) HEK293 cells were co-transfected with an expression vector of murine NFAT1, a firefly luciferase (Fluc) expression plasmid (pGL4.53[luc2/PGK]), and pNL3.2[NlucP/minP] containing different numbers (none, 3, 6 and 9) of the IL8 NFAT-RE. One day after transfection, cells were stimulated with ionomycin (IM, 1 μM; vehicle, 0.007% ethanol) for 6 h. Cell lysates were used to measure luminescent signals of NanoLuc (Nluc) and Fluc using a Nano-Glo Dual-Luciferase Reporter Assay System. The ratio of Nluc to Fluc, Nluc/Fluc, is expressed as normalized relative luciferase activity (RLA). Dots and bars represent individual and averaged RLA values obtained from triplicate assays, respectively. (B) HEK293 cells were co-transfected with expression plasmids for NanoLuc reporter and Fluc together with either wild type (WT), constitutively active type (CA) murine NFAT1 or empty vector (pcDNA3). One day after transfection, cells were pre-treated with FK506 (10 μM) or vehicle (0.16% ethanol) for 1 h and then subjected to ionomycin (IM) stimulation for 6 h, followed by luciferase assays.