Fig. 5
TMIGD1 directly interacts with BANF1 and modulates the NF-κB pathway. A Silver stain identified the TMIGD1-protein complex immunoprecipitated by anti-IgG or anti-FLAG antibody from the lysates of NCM460 cells transfected with lentivirus overexpressing FLAG-tagged TMIGD1. The arrowheads indicate bands of TMIGD1 and BANF1. B Proteomics analysis showed the 15 most enriched proteins from the immunoprecipitate of anti-FLAG antibody. C, D Immunoprecipitate by anti-FLAG antibody (C) or anti-BANF1 antibody (D) from the lysates of NCM460 cells transfected with lentivirus overexpressing FLAG-tagged TMIGD1. IP lysate (10%) was used as input. E GST pull-down assays of purified recombinant TMIGD1-GST and BANF1-Flag proteins. F, G Confocal microscope showed colocalization of TMIGD1 and BANF1 in NCM460 cells (F) and human epithelial mucosa (G). Scale bars, 20 μm for NCM460 cells and 100 μm for human epithelial mucosa. H Protein levels of BANF1, p65, and phosphorylated p65. I Protein levels of BANF1, p65, and phosphorylated p65 after TNF-α stimulation in NCM460 cells. J GSEA revealed significant enrichment of the NF-κB pathway. K Genes relevant to the NF-κB pathway were enriched in the comparison of WT+DSS and Tmigd1INT-KO+DSS groups according to KEGG analysis. L After TNF-α stimulation, the p65 protein level in cytoplasmic and nuclear fractions (down) and results of transcription factor binding assay of nuclear p65 (up) of NCM460 cells. Data are expressed as mean ± SEM. *** p<0.001