Figure 5

Inhibition of CRL–Nedd8 pathway globally affects cell signaling and transcription. (a) Dual luciferase reporter assay with VCaP cells co-transfected with control Renilla luciferase and Firefly luciferase reporter vectors (Table 1 and Supplementary Table S3). Firefly luciferase signal was first normalized to Renilla luciferase, and then to vehicle control (mean±S.D.). Responses were divided into; non-classified (NC, see Table 1); MLN^low- specific (low); MLN^high- specific (high). (b) MLN stabilizes cell signaling factors. Western blotting was performed with protein-specific antibodies. The protein level normalized first to GAPDH, then to vehicle control. (c) Effect of IKK inhibitors on MLN-induced apoptosis. Cells were treated for 5 d with indicated drugs in 10% ChSM. The data are presented as a boxplot diagram with P-values for 500 nM MLN. The four-point plot inside the diagram shows IKKβ-to-IKKα selectivity of the drugs.³⁴ See also Supplementary Figures S11–S13