Measuring NF-κB activity using a luciferase reporter system.

Cells were seeded in quadruplicate wells of 12-well tissue culture plates (Corning) in DMEM without antibiotics. The next day, the cells were transfected using 5 μl of Lipofectamine 2000 (Invitrogen) following the manufacturer's protocol. The amounts of DNA used for each transfection are indicated in the figure legends. For the IκB experiment (Fig. 4A), one experimental replicate used 20 ng of the IκB plasmids and the other used 40 ng IκB plasmid. For the MLN4924 experiment (Fig. 4C), one experimental replicate used 100 nM MLN4924 and the other used 500 nM MLN4924. Cells were harvested 24 hours after transfection. Two-thirds of the cells from each well were used for the Dual-Glo luciferase assay (Promega) according to the manufacturer's protocol. A SpectraMax M3 plate reader (Molecular Devices, Sunnyvale, CA, USA) was used to measure luminescence for the signaling experiments shown in Fig. 2 through 8. The Enspire multimode plate reader (PerkinElmer, Waltham, MA, USA) was used to measure luminescence for the signaling experiments shown in Fig. 1. Using both devices, firefly luciferase activity was measured and quenched first, and Renilla luciferase was measured subsequently. The remaining one-third of the cells from each of the wells was used for the detection of protein expression by WB.