Role of Nampt in modulating response to bortezomib. (A) MM1S cells were infected with either lentiviral construct expressing control scrambled or shRNA targeting Nampt. Total protein extracts were then subjected to immunoblot analysis with anti-Nampt or anti-GAPDH antibodies (left). The effect of Nampt knockdown on bortezomib response was assessed by measuring viability of infected cells (with shRNA scramble or targeting Nampt) after bortezomib treatment (1.25–5 nM) by using PI staining followed by FACS analysis (right). (B) Representative immunoblot images showing Nampt overexpressed in MM1S and U266 cells. Antitubulin monoclonal antibody served as loading control (top). Relative expression of Nampt protein was calculated by taking the ratio of the densitometry signal for Nampt to tubulin in each sample using Image J software (bottom). Cells overexpressing Nampt were subjected to bortezomib treatment of 24 h, and then viability was measured by MTT analysis (right). (C) U266 and MM1S cells were infected with a specific lentiviral shNampt or scramble control and then treated with 2 nM bortezomib for 24 hours. Thereafter, cells were used for cell lysates preparation, and Nampt, Mcl-1, bcl-2, and tubulin were detected by immunoblotting.