TNF induces NF-κB binding and IκB proteolysis. (A) TNF rapidly induces nuclear NF-κB Rel A DNA binding. U937 cells unstimulated or stimulated with 20 ng/mL TNF for the indicated times (at top). Cells were lysed, the nuclei were isolated, and subjected to EMSA analysis with a radiolabeled IL-8 NF-κB site. The bound complexes (C1-C3) are indicated. Unlabeled duplex wild-type (wt) or mutant NF-κB (▵κ) competitors were included where indicated. (B) Antibody interference. EMSA of TNF-stimulated nuclear extract was prepared. Either normal rabbit serum (NRS), anti-p50, or anti-Rel A antibodies were preincubated for 1 hour before the assay as indicated. Asterix is a faint supershifted band. C1 and C2 are completely attenuated by the Rel A antibody. Bottom: lighter exposure. C3, C2, and C1 are attenuated by the p50 antibody. (C) TNF induces time-dependent proteolysis of the IκB protein. Cells unstimulated/stimulated with 20 ng/mL TNF for the indicated times (top) were lysed and the cytosols were prepared. Top panel, Western immunoblot with anti-IκB antibody; bottom panel, Western immunoblot with an anti-β–actin antibody (internal control).