Defective nuclear translocation of the IRF8K108E mutant. (A) RAW macrophages stably expressing HA-tagged K108 and E108 variants were stimulated or not with IFN-γ (400 U/mL) for 3 hours. Cells were fixed, permeabilized, and incubated with mouse anti-HA mAb followed by mouse Cy3-conjugated secondary antibody (red) and then with DAPI to stain nuclei (blue). Cellular localization was analyzed by confocal microscopy using a Zeiss LSM5 Pascal laser scanning confocal microscope. All image analyses were performed using LSM5 Image. Two different images of each condition are shown. Images are representative of 3 independent experiments. Mean fluorescence intensity of Irf8 (red) staining was measured using Image J. Ratios of cytoplasmic over nuclear fluorescence are represented as means ± SD of 10 independent measurements. (B) RAW macrophages stably expressing HA-tagged K108, E108 were stimulated or not with IFN-γ (400 U/mL) for 3 hours. Cell lysates were subjected to cellular fractionation to separate the cytoplasmic and nuclear fractions and immunoblotted with anti-IRF8, anti-RNA Pol II (nuclear control), and anti-GAPDH (cytoplasmic control) antibodies. The white triangle represents transfected E108, and the black triangle represents endogenous K108. The relative nuclear and cytoplasmic distribution of the total K108 and E108 proteins was evaluated and is shown.
