Prl cmv reference plasmid

24-well plates were seeded with 3 × 10⁵ HEK293T cells per well. The day after, cells were transfected with the empty pCI-neo-3xFlag expression vector or encoding viral proteins as specified. Cells were jointly co-transfected with 100 ng of firefly luciferase constructs under the control of the IFN-β or the ISG56 promoter and 10 ng of the pRL-CMV reference plasmid (Promega, Madison, WI, USA). Finally, 300 ng of pNRIG-I was transfected to activate the indicated promoters. The total amount of DNA was equally adjusted with pCI-neo-3xFlag. After 40 h, transfected cells were collected, and both firefly and Renilla luciferase activities were determined using the Bright-Glo and Renilla-Glo luciferase assay system (Promega), respectively. All graphs represent mean ratios between luciferase and Renilla of triplicate samples and include error bars of the standard deviation.

HEK-293T were plated in 24-well plates (5 × 10⁵ cells per well). One day later, HEK-293T cells were co-transfected (JetPRIME; Polyplus) with IFN-β-pGL3 or pISRE-Luc plasmid (0.3 μg/well; Stratagene) that contain the firefly luciferase reporter gene downstream of an IFN-β-specific promoter sequence or the ISRE enhancer element upstream, respectively. Cells were simultaneously co-transfected with the pRL-CMV reference plasmid (0.03 μg/well; Promega) and the empty pCI-neo-3×FLAG expression vector (0.3 μg/well) or encoding proteins as specified. When specified, cells were transfected with 0.1 μg/well of poly(dA:dT) (Invivogen) or treated with 1 x 10³ IU/ml of recombinant IFN-β (PBL Assay Science) 24h after transfection. After 24h post-IFN-β-stimulation or 48h post-transfection, the cells were lysed (Passive lysis buffer, Promega), and both firefly and Renilla luciferase activities in the lysate were determined using the Bright-Glo and Renilla-Glo luciferase assay systems (Promega), respectively. The reporter activity was calculated as the ratio of firefly luciferase activity to the reference Renilla luciferase activity. All graphs show mean values and include error bars indicating the standard deviations (SD).