Fig. 5. The role of NPM1 in p65 signaling.
a, Schematic diagram of protein–protein interaction analysis with NPM1. b, Silver-stained gel showing proteins that were immunoprecipitated with NPM1 and exhibited higher intensity in stimulated than unstimulated cells. Red rectangle shows bands that were excised for MS analysis. c, Top five candidate NPM1-interacting proteins identified by MS. d, IP and IB of the interaction between NPM1 and p65 in MNK3 cells. The samples were derived from the same experiment, and the blots were processed in parallel. e, Immunofluorescence staining of the subcellular location of p65 in ILC3s isolated from mice at day 5 of administration of 2.5% DSS or under the steady state (water). Scale bar = 5 μm. f, Unstimulated or stimulated MNK3 cells were subjected to cellular fractionation into cyto and nuc fractions followed by western blotting for p65. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and histone H3 were used as markers for cytosolic and nuclear proteins, respectively. g, RT–PCR analysis of mRNA abundance of p65 target genes in isolated colonic ILC3s from Npm1+/+ and Npm1+/− mice at day 5 of administration of 2.5% DSS (n = 5 individual mice). h, KEGG pathway enrichment analysis of upregulated transcription factor-related pathways in ILC3s from patients with UC compared with ILC3s from healthy participants. i, Gene Ontology (GO) analysis of downregulated pathways in NPM1low ILC3s compared with NPM1high ILC3s from patients with UC, which was identified using a median expression cutoff for NPM1 in ILC3 of patients with UC. j–m, Cell mito stress test was performed with stimulated MNK3 cell line with or without p65 knockdown. Representative OCR (j), basal OCR (k), ATP production (l) and maximal respiration (m) are shown (n = 3 biological samples). n, Expression of Il22 in unstimulated and stimulated MNK3 cell line (shNC and shp65; n = 3 biological samples). Data in g and k–n are representative of two independent experiments, shown as the means ± s.e.m., and statistical significance was determined by two-tailed unpaired Student’s t test (*P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001). IB, immunoblot; cyto, cytosol; nuc, nuclear.