IKKβ inhibits Stat-1 activation in GBS-infected macrophages. (A) Peritoneal macrophages from IKKβ^ΔMye and IKKβ^f/f mice were stimulated in vitro with heat-killed GBS (MOI of 20:1). Protein extracts were prepared at the indicated time points for biochemical analysis. IKK and JNK activity was measured by IP kinase assay (KA) using recombinant substrates GST-IκBα^1-54 or GST-cJun^1-79, respectively. NF-κB activation was measured by electrophoretic mobility shift assay (EMSA) using ³²P-labeled κB consensus oligonucleotide. (B) Expression of IKK, pro–IL-1β, NOS2, tyrosine 701 phosphorylation of Stat1 (pY-Stat1), serine 726 phosphorylation of Stat1 (pS-Stat1), total Stat1, and SOCS1 was measured by immunoblot analysis of cell lysates using actin as a loading control. (C) In parallel experiments, TNF-α production in cell culture supernatants was measured by ELISA, and (D) total RNA was isolated for real-time PCR analysis of TNF-α (Tnfa) and IFN-β (Ifnb) mRNA expression. Data are represented as mean ± SEM of n = 4. Representative data are shown from at least three independent experiments.