Fig. 3.

ISG15 secretion and the induction of IFN-γ production in leukocytes. Control and P1 leukocytes were left unstimulated or were stimulated with BCG and IFN-α2b. After 0 h and 12 h, cells and supernatants were harvested and subjected to western blotting (A) or TNF-α ELISA (B). Resting control and P1 leukocytes were labeled extracellularly with CD16 and CD3 antibodies and intracellularly with ISG15 antibody and were subjected to ImageStreamX analysis with bright field (BF) and side scatter (SSC) also shown (C). IFN-γ secretion was measured by ELISA in PBMCs stimulated with vehicle (−), various doses of recombinant human ISG15 (including boiled recombinant human ISG15 to exclude LPS contamination) (D), recombinant human ISG15 and IL-12, alone and in combination (E). The IFN-γ secretion results shown are representative means of at least 3 independent experiments. Error bars indicate the SEM.