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Previous studies demonstrated that induction of the heat shock response is associated with inhibition of the proinflammatory transcription factor NF-κB by a mechanism involving inhibition of I-κBα degradation. To provide further insight regarding the interactions of these fundamental cellular responses, the present experiments were designed to elucidate the mechanism(s) by which heat shock inhibits degradation of I-κBα. In an in vitro model of inflammatory cell signaling, treatment of RAW 264.7 murine macrophages with LPS (100 ng/mL) caused rapid degradation of I-κBα. Heat shock, 1 h before treatment with LPS, completely inhibited LPS-mediated degradation of I-κBα. Immunoprecipitation studies demonstrated that heat shock inhibited LPS-mediated ubiquitination of I-κBα. Western-blot analyses using a phosphorylated I-κBα-specific antibody demonstrated that heat shock inhibited LPS-mediated phosphorylation of I-κBα. In contrast, heat shock induced phosphorylation of c-jun. In murine fibroblasts having genetic ablation of the heat shock factor-1 gene, heat shock inhibited tumor necrosis factor-α mediated degradation of I-κBα. We conclude that the mechanism by which heat shock inhibits LPS-mediated degradation of I-κBα involves specific inhibition of I-κBα phosphorylation and subsequent I-κBα ubiquitination. In addition, this mechanism does not involve activation of heat shock factor-1 or the heat shock proteins regulated by heat shock factor-1.