Effects of NPI-0052 plus bortezomib on Hsp's and proteasomal activities. (A) MM.1S cells were treated with NPI-0052 (1 nM), bortezomib (3 nM), or combined NPI-0052 (1 nM) plus bortezomib (3 nM) for 12 hours and harvested; total proteins were then subjected to immunoblot analysis with anti–phospho-JNK or anti–actin Abs. Blots shown are representative of 3 independent experiments. (B) MM.1S cells were treated with NPI-0052 (1 nM), bortezomib (3 nM), or combined NPI-0052 (1 nM) plus bortezomib (3 nM) for 12 hours and harvested; total proteins were then subjected to immunoblot analysis with anti–phospho-eIF2-α, anti–CHOP/GADD153, or anti–actin Abs. Blots shown are representative of 3 independent experiments. (C) MM.1S cells were treated with NPI-0052 (1 nM), bortezomib (3 nM), or combined NPI-0052 (1 nM) plus bortezomib (3 nM) for 24, 48, and 72 hours and harvested; total proteins were then subjected to immunoblot analysis with anti–Hsp-90, anti–Hsp-27, or anti–Hsp-70 Abs. Lysates from HeLA cells served a positive control in immunobloting with Hsp Abs. Blots shown are representative of 2 independent experiments. (D) MM.1S cells were treated with NPI-0052 (1 nM), bortezomib (3 nM), or combined NPI-0052 (1 nM) plus bortezomib (3 nM) for 24 hours and harvested; nuclear extracts were then analyzed for NF-κB activity by using a p65 enzyme-linked immunosorbent assay (ELISA) kit (Active Motif, Carlsbad, CA). (E) MM.1S cells were treated with indicated concentrations of NPI-0052, bortezomib, or combined NPI-0052 plus bortezomib for 30 minutes and harvested; cytosolic extracts were then analyzed for CT-L, C-L, and T-L proteasomal activities. The data are represented as percentage of inhibition compared with vehicle control. Data are presented as means plus or minus SD. (n = 3; P < .05).