2.7. NF-kB Luciferase Reporter Assay

HEK293t cells were cultured in DMEM media supplemented with 10% FBS, 1% penicillin/streptomycin, and 0.1 mM MEM non-essential amino acids (Invitrogen). In black, flat-bottomed tissue culture 96-well plates (Corning, Glendale, AZ, USA), NFKBIE expression vectors (0.4 ug), pGL4.32(luc2P/NF-kB-RE/Hygro) reporter vector (2.0 ug, Promega, Madison, WI, USA), and pRL-TK vector (0.2 ug, an internal transfection control, Promega) were transfected into HEK293t cells using Lipofectamine 2000 (Life Technologies). After 48 h, the cells were incubated with 10 ng/mL TNFalpha (Gemini Bio, Sacramento, CA, USA) for 4 h or media only. Reporter activity was measured using the Dual-Glo Luciferase assay system (Promega) and read on the Synergy 2 (BioTek, Winooski, VT, USA).