Bortezomib impairs TLR signaling cascades in DCs. Peripheral blood adherent monocytes were cultured with GM-CSF and IL-4 for 6 days. Subsequently, cells were incubated with the indicated concentrations of bortezomib (in ng/mL) for 8 hours before 100 ng/mL LPS was added to the culture medium. Sixteen hours later, cells were harvested and used for nuclear extracts (A) or whole-cell lysate (B-C) preparation. Nuclear-localized RelB, IRF-3, and IRF-8 (A) and the phosphorylation state of ERK and p38 (B) as well as MyD88 levels (C) were detected by immunoblotting. Equal protein loading was confirmed by staining with ponceau S. (D) RNA was isolated from DCs that were obtained from adherent monocytes by culture with GM-CSF and IL-4 and thereafter stimulated with LPS with or without bortezomib as in panel A. Thereafter, cDNA was synthesized and used for semiquantitative PCR amplification of TLR4 and β2m cDNA.