Knockout of IκBβ exhibit reduced IL-1β expression in macrophages (BMDMs). (A) WT and IκBβ^−/− BMDM (three animals for each group) were stimulated with LPS (1 µg/ml) as indicated. Total RNA was prepared and IL-1β, TNF, and IL-6 mRNA levels were quantified using real-time PCR analysis (left; Student’s t test; *, P < 0.01 versus control). IL-1β, IL-6, and TNF cytokine secretion in response to LPS was determined by ELISA (right; Student’s t test; *, P < 0.01 versus control). Results are shown as the mean of two independent experiments. (B) BMDMs from WT and IκBβ^−/− mice were stimulated with LPS (1 µg/ml) for indicated time points or were left as an untreated control. Western blot detects IκBβ and pro–IL-1β expression. Similar results were obtained from two additional experiments. β-actin was used as loading control. (C) EMSA using a radiolabeled probe containing an NF-κB–binding site. BMDMs were LPS-treated (100 ng/ml) as indicated and nuclear extracts were analyzed by EMSA. An Oct-consensus oligonucleotide was used to control equal protein input. Three independent experiments revealed similar results. (D) BMDMs isolated from WT and IκBβ^−/− mice were stimulated with LPS (100 ng/ml) and analyzed by Western blot. Expression levels of indicated NF-κB proteins and IκB members were determined at the indicated time points. One out of three independent experiments is shown.