Pgl nf κb luc

The luciferase reporter plasmids pGL-NF-κB-Luc (10 μg) and pGL-TK-Luc (10 μg) (Promega) were electroporated (250 V, 950 μF) into Jurkat cells (5 × 10⁶). After 48 h, cells were collected and stimulated with murine recombinant TNFα (20 ng/ml, R&D Systems) or anti-CD3ε/CD28 antibodies (1 μg/ml, BioLegend) for the indicated times. Next, the cells were lysed and ligand-dependent NF-κB activity was measured (in terms of luciferase activity) using the Dual-Luciferase Reporter or Bright-Glo luciferase assay system (Promega), according to the manufacturer’s protocol. Luminescence was detected in a Lumat luminometer (Berthold).

Dual-luciferase reporter assays to detect the effects of ccIRF4 on the activation of NF-κB were performed in 293 T cells by using pIRF4, pMyD88 or empty vector together with a luciferase-linked NF-κB. Transfection assays were performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. The 293 T cells in 96-well plates were transfected with reporter gene plasmids, pGL-NF-κB-Luc, pGL-Renilla-luc plasmid (Promega), and the correct amount of expression plasmids or empty expression vectors (as control). The pGL-Renilla-luci plasmid was used as internal control. At 48 h post transfection, the Dual-Glo® Luciferase Reagent (Promega) was used to measure the activity of firefly and Renilla luciferase according to the manufacturer’s instructions with each experiment done in triplicates.