TNF-a activates NF-kB in RAW264.7 cells. A. TNF-a promotes degradation of IkBa and resynthesis over 60 minutes. IκBα and β-actin protein levels were assessed by Western blot analysis and quantified by densitometry (lower panel). Densitometry data show IκBα levels that were corrected by β-actin and represent normalised data from four independent experiments. * = P < 0.05. B. EMSA for NF-kB proteins in nuclear extracts of RAW264.7 cells over six hours of TNF-a exposure. The arrow on the left indicates the position of the RelA/NF-kB1 heterodimer. Competition for binding by an unlabeled specific oligonucleotide (lane 7) but not by an irrelevant sequence at 100-fold molar excess (lane 6) confirmed specificity of DNA binding. Supershift analyses with specific antibodies to RelA, NF-kB1 (p50), c-Rel and RelB (lanes 9, 10, 11 and 12, respectively, of the right panel) confirmed the identities of RelA and NF-kB1 in the complex with a lighter supershift c-Rel band. Arrows on the right indicate the locations of supershifted bands. The figure is representative of two independent experiments. C. TNF-a (10 ng/ml) induces NF-kB activity, as estimated by luciferase activity in RAW264.7 cells that had been transiently transfected with the pNF-kB-TA-Luc reporter. Results are representative of three independent experiments.