Figure 5.

NMI inhibits the nuclear translocation of NF-κB/p65 after TNF-α stimulation. (A) HeLa cells were transfected with control or Myc-NMI plasmids, and at 24 h posttransfection, the cells were left untreated or treated with TNF-α (10 ng/ml) for 60 min. The cells were fixed and incubated with anti-p65 or anti-Myc Ab and then stained with rhodamine-conjugated anti-mouse IgG (red) or FITC-conjugated anti-rabbit Ab (green). The same slide was also stained with DAPI to show the nucleus. The expression and localization of p65 and Myc-NMI was determined by confocal immunofluorescence analysis. The percentage of the cells expressing p65 in the nucleus among the cells that express Myc-NMI and the cells that don’t express Myc-NMI was calculated in the bottom panel. (B) HeLa cells were transfected with control or Myc-NMI plasmids, and at 24 h posttransfection, the cells were left untreated or treated with TNF-α (10 ng/ml) for 60 min. The cells were then harvested and fractionated into the nuclear and cytoplasmic fractions, and the fractions were immunoblotted with anti-NMI and anti-p65 Abs. The quantification by densitometry of western blots of p65 bands without or with Myc-NMI in TNF-α-stimulated nuclear fractions was shown in the right panel. The results are representative of three independent experiments, and the error bars represent the SD.**p < 0.01. (C) HeLa shCtrl and HeLa shNMI cells were treated in (B), and performed the cellular fractionation assay.