Fig. 4.

Nuclear-cytoplasmic partitioning of NIBP is regulated by RNF138 and contributes to the activation of NF-κB signaling. a Co-immunoprecipitation analysis of endogenous RNF138 and NIBP interactions in HCT116 total lysates (left) and nuclear extracts (right). IgG was used as control. b Immunostaining analysis of RNF138-NIBP co-localization in HCT116 and RKO cells. Nuclei were stained with DAPI. Insets show stained areas in more detail. Scale bar, 20 μm. c Representative confocal images (left) and quantification (right, n = 20) of endogenous RNF138 and NIBP interactions in HCT116 and RKO cells using proximity ligation assay. IgG were used as controls. Scale bar, 5 μm. d Immunostaining analysis of the co-localization of endogenous IKKβ and NIBP in HCT116 and RKO cells. Scale bar, 5 μm. e Immunoblots of phosphorylation, total levels of p65 in HCT116 and RKO cells transfected with si-NIBP or control siRNA. f qPCR analysis of NF-κB target gene expression in HCT116 and RKO cells (n = 3) with NIBP inhibition. *P < 0.05; **P < 0.01 and ****P < 0.0001. g Immunoblots of phosphorylation and total levels of p65 and IKKβ in RNF138^WT and RNF138^KO HCT116 and RKO cells with NIBP inhibition. h Representative immunostaining images of NIBP localization in RNF138^WT and RNF138^KO HCT116 and RKO cells, and the distribution of relative fluorescence intensities of lines scanned across the nucleus (right). Quantification of cytoplasmic NIBP intensity in HCT116 and RKO cells (n = 20). Scale bar, 5 μm. i Immunostaining analysis of IKKβ-NIBP co-localization in RNF138^WT and RNF138^KO HCT116 and RKO cells and relative pixel intensity of lines scanned across the nucleus. Scale bar, 20 μm