Interactions between IκBs and p65 in CLL. (A) Co-IP to study the interaction between p65 and IκBε in NFKBIE-deleted (n = 3) versus WT (n = 2) CLL. The bottom panel indicates TCL from samples used in the co-IP assay. (B) Mean values for IκBε pull-down in NFKBIE-deleted versus WT CLL. (C) Proximity ligation assay to study the physical interaction between IκBα, IκBβ and IκBε, and p65 in six NFKBIE WT CLL patients. Interactions were assessed in unstimulated (U), αIgM-stimulated, and CD40L-stimulated cells. For IκBα and IκBε, six NFKBIE WT CLL patients were analyzed, whereas for IκBβ only four NFKBIE WT CLL patients were assessed. (D) The interaction between IκBε and the transcription factor p65 in six NFKBIE WT CLL patients and six NFKBIE-deleted patients. Interactions were again assessed in unstimulated (U), αIgM-stimulated, and CD40L-stimulated cells. Error bars indicate standard error. (E and F) Fluorescent microscope images of the interaction between IκBε and the transcription factor p65 in cells from a NFKBIE WT CLL patient (E) and an NFKBIE-deleted CLL patient (F). Blue color indicates cell nuclei, whereas each red dot represents a single interaction. Bars, 20 µm.