IL-1β-activated protein complexes contain NF-κB p65 and p50 factors.

(A) Competition EMSA. Nuclear extracts were prepared from rat primary hepatocytes co-stimulated with IL-1β and IL-6. The different probes used in competition EMSAs are indicated as: Site II, γ fibrinogen site II probe; SIE, mutant 67 of the serum inducible element onc-fos gene promoter, which has a strong binding affinity for STAT3; NF-κB, NF-κB binding site on the immunoglobulin κ chain enhancer; C/EBP, CAAT-enhancer binding protein binding site on the IL-6 gene promoter. Competition EMSAs and the DNA sequences of these probes are described in “Materials and methods.” (B) Analyses of IL-1-induced protein complexes with antibodies and IκBα protein. Nuclear extracts were prepared from rat primary hepatocytes stimulated with IL-1β. The antibodies and reagents are indicated. The supershift assays were performed as described in “Materials and methods.” For IκBα treatment, nuclear extract was preincubated with 1 ng of recombinant IκBα prior to EMSA. (C) Cross-competition EMSA. Radio-labeled α2MG probe was used in the binding assay together with the indicated molar excess of unlabeled site I, site II, and site III probes for cross-competition. The arrows on the left indicate the migration position of STAT3 and NF-κB complexes.