Inhibition of CT-L subunits is sufficient to cause modulation of polyubiquitinated proteins, Noxa and phospho-eIF2α. (A) MM1.S cells were 1-hour pulse-treated with DMSO, 0.18 μM CPSI (c), 0.27 μM IPSI (i), 0.18 μM CPSI plus 0.27 μM IPSI (c + i), 20 nM carfilzomib (CFZ 20 nM), or 10 μM carfilzomib (CFZ 10 μΜ), washed and incubated in compound-free media for a total of 4 hours. Cell lysates were electophoresed and transferred for Western blot detection with antibodies targeted against polyubiquitin, actin, Noxa, and IκBα. Quantitation of polyubiquitinated proteins (B) and Noxa (C) in compound-treated MM1.S cells. Mean ± SD for 3 independent experiments are shown. (D) MM1.S cells were 1-hour pulse-treated with 0.27 μM IPSI (i), 0.18 μM CPSI (c), 0.18 μM CPSI plus 0.27 μM IPSI (c + i), 20 nM carfilzomib (CFZ 20 nM), or 10 μM carfilzomib (CFZ 10 μΜ), DMSO, 1 μM brefeldin A (BfA), 1 μM thapsigargin (TG), and 2.5 μg/mL tunicamycin (TU), washed and incubated in compound-free media for a total of 6 hours (brefeldin A, tunicamycin, and thapsigargin were added back to the media). Cell lysates were electophoresed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred for Western blot detection with antibodies targeting CHOP, eIF2α, phospho-eIF2α, and actin. (E) Quantitation of phospho-eIF2α in compound-treated MM1.S. Mean ± SD of 3 independent experiments are shown. One-way analysis of variance was performed and compound treatments resulting in statistically significant differences (as determined by Bonferroni post-hoc testing): *P < .05, **P < .01, ***P < .001.