Distribution of IFIT4 in tissues and cells, and the effect of IFN-α on IFIT4 expression. Using real-time quantitative RT-PCR, IFIT4 mRNA relative expression was determined among (a) 14 normal human tissues and (b) four kinds of immune cells. To determine the subcellular location of IFIT4 protein, THP-1 cells were transfected with (c) (subpanel a) pEGFP-C1 control or (subpanel b) pEGFP-IFIT4 plasmid. Forty-eight hours later, cells were stained with DAPI for nuclear staining and examined by confocal microscopy. The effect of IFN-α2a on the localization of IFIT4 protein was also observed. (c) (subpanel c) THP-1 cells transfected with pEGFP-IFIT4 were further stimulated with 3,000 μ/ml IFN-α2a for 72 hours. Blue colour shows the location of the nucleus, whereas green colour shows the sublocalization of green fluorescent protein alone or fused with IFIT4 protein. (d) To analyze the effect of IFN-α on the expression level of IFIT4, peripheral blood mononuclear cells (PBMCs) from healthy donors were treated with 3,000 μ/ml IFN-α2a for 24 hours, 48 hours or 72 hours, and then IFIT4 mRNA in the PBMCs was detected by real-time quatitative RT-PCR; all experiments were repeated three times with similar results. IFIT4 protein levels from normal PBMCs treated with IFN-α2a for 72 hours was examined by (e) flow cytometry with intracellular staining or (f) Western blotting with β-actin as a protein loading control. The experiments were performed three times and a set of representative histograms and data is presented. IFIT4, interferon-induced protein with tetratricopeptide repeats 4.