Fig. 1

IκB kinase α (IKKα) directly interacts with c-Myc. a Schematic illustration of IKKα and c-Myc domains: NBD: NEMO binding domain; ULD: ubiquitin-like domain; MBI – MBIV: Myc boxes; NLS: nuclear localization signal (dark blue); HLH: helix-loop-helix domain; LZ: leucine zipper. Positions of amino acids are indicated by numbers. b 3D- models of an IKKα homodimer (PDB-code: 5TQW) and a c-Myc-Max heterodimer bound to DNA (PDB-code: 1NKP), depicting comparable protein-interaction domains at the coiled HLH/LZ regions. c Interaction between IKKα and c-Myc was shown by co-immunoprecipitation. Either IKKα or IKKβ were immunoprecipitated (IP) from DU145 cell extracts (input), followed by SDS-PAGE and Western blot (WB) to detect c-Myc. d Verification of the IKKα/c-Myc interaction by FRET microscopy: Expression constructs of an EGFP-mCherry fusion protein (positive control, fusion), unbound EGFP and mCherry (negative control, separate proteins), EGFP-tagged c-Myc and mCherry-tagged IKKα fusion proteins, were transfected into HEK-293 cells as indicated. 1d after transfection FRET microscopy was performed as described in detail in the Methods section. Donor channel: EGFP excitation and emission); acceptor channel: mCherry excitation and emission; raw FRET channel: EGFP excitation and mCherry emission. The corrected FRET images show the FRET signal after subtraction of the spectral bleed-through (according to Youvan et al. [30]) and is a qualitative assessment of the interaction without normalization to expression levels). e Quantitative assessment of normalized FRET values based on corrected donor fluorescence (DFRET) as described in [26]. Statistical analysis was performed using two-way ANOVA and Dunnett’s multiple comparison, p-values: ‘****’ for p < 0.0001. Error bars represent mean ± standard deviation (n = 508 for the fusion control; n = 236 for the separate control and n = 394 for c-Myc + IKKα)