TNF-α-stimulated NF-κB activation assay.

<p>(A) and (B) Measurement of bioluminescence from cells without or with TNF-αstimulation. Monoclonal HeLa cells stably transfected with the NF-κB-secNluc-FLAG gene were inoculated at different densities (0 to 8000 cells per well) in 24-well plates for 12 h. For TNF-α stimulation, 50 ng/ml TNF-α was added for 12 h to induce the activation of the NF-κB signaling pathway (B). 100 μl of each cultured medium was subjected to magnetic bead purification, and the beads were resuspended in 100 μl of PBST. 100 μl of resuspended solution (Beads Purification), 100 μl of cultured medium (Medium), 50 and 10 μl of culture media that were supplemented with PBST to 100 μl (50% Medium and 10% Medium) were used to measure the emitted bioluminescence. The experiments were repeated three times with comparable results. (C) The ratios of RLU in groups with TNF-α stimulation to those in groups without TNF-α stimulation. The data shown are the means ± standard errors of three independent experiments.