Bortezomib triggers IKKβ phosphorylation; conversely, inhibition of IKKβ blocks bortezomib-induced IκBα down-regulation and NF-κB activation. (A) RPMI 8226 cells were cultured with bortezomib (20 nM) or MG132 (MG, 0.5 μM). TNFα (TNF, 5 ng/mL) served as a positive control for p-RIP2 and p-IKKβ. (B) MM.1S, U266, and OPM1 MM cell lines, as well as primary tumor cells from MM patients, were cultured with bortezomib (20 nM for 8 hours). (C) MM.1S, RPMI 8226, and U266 cells were cultured with bortezomib (10 nM and 20 nM) for 8 hours. (D) RPMI 8226 cells were cultured with bortezomib (20 nM) for 8 hours in the presence (5 and 10 μM) or absence of MLN120B (MLN). Whole-cell lysates and nuclear extracts were immunoblotted with indicated Abs and EMSA, respectively. RPMI 8226 cells (E) and primary tumor cells (F) from MM patients (no. 1 and 2) were cultured with bortezomib (10 nM) for 24 hours, in the presence (5 μM, ; 10 μM, ■) or absence (□) of MLN120B, and cell growth was assessed by MTT assay. * P < .01.