Fig. 4

IKKα, but not IKKβ increases the transcriptional activity of c-Myc and dependence of c-Myc stability on the IKK-target site. Transcriptional activity of c-Myc measured with a Nano-luciferase (Nanoluc) reporter gene construct under the control of the c-Myc dependent CDK4 promoter in DU145 cells. a Co-transfection of cells with expression constructs of IKKα or IKKβ as indicated; n = 5. b Transcriptional activity of a phosphorylation-deficient c-Myc mutant (c-MycAA) and a phosphomimicking c-Myc mutant (c-MycEE) in presence or absence of the IKK-inhibitor BMS-345541; n = 4. c c-Myc activity in control-, IKKα-, IKKβ- and c-Myc knockout cells upon treatment with DMSO (control) or BMS-345541; n = 4. d Stable DU145 transfectants expressing c-MycAA or c-MycEE were cultured for different time periods in presence of 60 μg/ml cycloheximide to block protein synthesis, followed by cell lysis, SDS-PAGE and Western blotting for c-Myc. The c-Myc-specific bands were quantified by ImageJ and subject to single exponential decay curve fitting. Statistical analysis was performed with GraphPad Prism using a: one-way ANOVA and Tukey’s post hoc test, (b, c): two-way ANOVA and Tukey’s multiple comparison and (d): curve fitting with a one-phase exponential decay equation using a constraint of 100% as initial value and a constraint of 0% for the plateau. p-values: ‘*’ for p < 0.05; ‘***’ for p < 0.001; ‘****’ for p < 0.0001. Mean values are plotted with error bars indicating ± standard deviation