Fig. 6
DDX27 promoted the interaction between NPM1 and p65 in nucleus and increased the binding activity of p65. a 293 T cells were transfected with pcDNA3.1-DDX27-BirA-HA or empty vector. Cells were incubated with 50 uM biotin and lysates were immunoblotted with indicated antibodies. 293 T cell lysates transfected with pcDNA3.1-DDX27-BirA-HA were pulled down by streptavidin beads and target proteins of DDX27 were identified by subsequent affinity purification mass spectrometry. b 293 T cell lysates transfected with pcDNA3.1-DDX27-BirA-HA were pulled down by streptavidin beads and immunoblotted with indicated antibodies. c The interactions between DDX27 and NPM1 were verified in HCT116 and SW480 with endogenous DDX27 immunoprecipitation and endogenous NPM1 immunoprecipitation, respectively. d In situ proximity ligation assay (PLA) detection of endogenous p65 and NPM1 interaction. HCT116 cells were incubated with or without TNF-α for 2 h. After fixation, in situ PLA for NPM1 and p65 was performed with NPM1 and p65-specific antibodies. The red dots (PLA signal) indicate an interaction. The nuclei were counterstained with DAPI (blue). The number of PLA dots in nucleus per cells was determined (at least 5 different regions (Obj: X63) were counted for each group). Mann–Whitney U-test was performed. e HCT116 and SW480 cells were treated with 50 ng/ml TNF-α for 2 h. In DDX27-overexpressing cells, enhanced protein interaction between NPM1 and NF-кB p65 was exhibited by endogenous p65 immunoprecipitation. f Recruitment of p65 to its target genes. HCT116 cells were incubated with or without TNF-α for 2 h, followed by ChIP assay. The precipitated DNA amounts containing the promoter regions of the genes (BIRC3 and TNF-α) by DDX27 overexpression were quantitatively analyzed by qPCR. Student’s t-test was performed. (*P < 0.05; ***P < 0.001)
