ICSBP and IRF-4/Pip form a complex on the ISG15 ISRE. (A) ICSBP, IRF-2, and IRF-4/Pip form a complex on the ISG15 ISRE in B lymphocytes. Mobility shift assay with the ISG15 ISRE probe and B-lymphocyte nuclear extracts from wild-type (lanes 1 to 5) and ICSBP-deficient mice (lanes 6 to 10). Lanes 1 and 6, competition with unlabelled ISG15 ISRE oligonucleotide; lanes 3 and 8, anti-ICSBP antibody; lanes 4 and 9, anti-IRF–2 antibody; lanes 5 and 10, anti-IRF–4/Pip antibody. The position of the bands, B1, 2, and 3, formed on the ISG15 ISRE are indicated with arrows. (B) IRF-4/Pip is present in macrophages and forms a complex with ICSBP. Mobility shift assay showing complex formation of the ISG15 ISRE and proteins from primary macrophage nuclear extracts from wild-type (lanes 1 to 3) or ICSBP-deficient mice (lanes 4 to 6). Lanes 3 and 6, anti-ICSBP antibody; lanes 2 and 5, anti-IRF–4/Pip antibody. The arrow shows the specific complex binding to the ISG15 ISRE. The slowly migrating bands seen in the ICSBP−/− extracts are nonspecific bands. (C) Western blot indicating the presence of IRF-2 in mouse macrophages.