Pevonedistat Modulates Cell Signaling

The CLBL-1 cells were treated with DMSO control or pevonedistat (0.1–0.7 μM) for 1, 4, 6, 12 or 24 hours. Cell lysates were made according to the previously published methods^{16 (link)} and were described in detail in the supplementary data. For detection of p-IκBα, CLBL-1 cells were stimulated with 10 ng/mL of human recombinant (rh) TNFα (PeproTech, Rocky Hill, NJ) for 5 minutes before preparing cell lysates in both pevonedistat and DMSO-treated groups. For detection of cleaved Caspase 3, and phosphorylated Histone 2AX, cells were not stimulated with any cytokine. The following commercial antibodies were used in western blots: p-IκBα (1:1000, Cell Signalling Technology, Danvers, MA, 14D4), cleaved caspase-3 (1:1000, Cell Signalling Technology, Danvers, MA, 5A1E) and p-H2AX (1:1000, Santa Cruz Biotechnology, Dallas, TX, sc-517348), and β-actin (1:5000, Sigma-Aldrich, AC-15).