The human myeloma cell line U266 was purchased from American Type Culture Collections (Rockville, MD). Myeloma cells were routinely maintained in RPMI 1640 supplemented with 1% penicillin/streptomycin, 1 mmol/L L-glutamine, and 10% fetal bovine serum at 37uC, 5% CO 2 in air. Bortezomib was provided by Millennium Pharmaceuticals (Cambridge, MA). To differentially label bortezomib-treated and -untreated U266 cells, the SILAC Protein Quantitation Kit (Pierce Biotechnology, Rockford, USA) was used according to the manufacturer's instruction. In brief, cells were grown in SILAC RPMI 1640 Medium (Pierce Biotechnology, Rockford, USA) containing 10% v/v dialyzed FBS, and either 0.1 mg/mL heavy [ 13 C 6 ] or light [ 12 C 6 ] L-lysine (Pierce Biotechnology, Rockford, USA). To ensure full incorporation of the heavy and light labeled amino acids, cells were grown for at least six cell doublings prior to analysis. U266 cells were treated with 3 nM bortezomib for 24 h, according to the half-maximal inhibitory concentration (IC50) measured by Hideshima et al [11] . After treatment, cells were washed three times with ice-cold washing buffer (10 mM Tris-HCl, 250 mM sucrose, pH 7.0) and transferred to a clean 1.5 mL Eppendorf tube. Cells were lysed with RIPA lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% SDS, 1% NP-40, 0.5% sodium deoxycholate, 1 mM PMSF, 100 mM leupeptin, and 2 mg/mL aprotinin, pH 8.0). Cellular debris was removed by centrifugation for 30 min at 13, 200 g and at 4uC. Protein concentrations were measured in duplicate using RC DC protein assay (BioRad, Hercules, CA, USA) and confirmed by SDS-PAGE.