Figure 5. ISG15 mRNA expression and intracellular protein production in PBMCs and cell subsets.

PBMCs were separated into monocytes (Mono), lymphocytes (Lympho), and an NK-enriched cell population (NK), using magnetic beads. (A) For ISG15 mRNA expression, 2×10⁶ cells/ml were cultured for 4, 8, and 20 hrs in the presence of IFN-α (IFN-α-treated cells) at 1,000 U/ml or FHA-2 (FHA-treated cells) at 5 µg/ml or without treatment. At each time point, total RNA was extracted and analyzed by RT-PCR. The fold expression change shown in the Figure is relative to levels in untreated cells at the beginning of the time course; the standard deviations for duplicate measurements are displayed. (B) For intracellular detection of ISG15, monocytes, NK-enriched cells, and lymphocytes were stimulated for 4 hrs with IFN-α (IFN-α-treated cells) at 1,000 U/ml or FHA-2 (FHA-treated cells) at 5 µg/ml or without treatment. Cells were then stained for intracellular ISG15 using the mouse monoclonal anti-ISG15 clone 4.1 labeled with AlexaFluor 647 (AF647). The number in the upper left corner of each graph represents the percentage of total cells testing positive for ISG15.