FIG. 7.

V12 IRF-5 is constitutively nuclear and negatively regulates IRF-5 transactivation. (A) V12 encodes a truncated IRF-5 protein. Western blot probed with anti-HA antibody comparing the electrophoretic mobility of polyacrylamide gel electrophoresis-separated, HA-tagged, in vitro-translated V3, V5, and V12 IRF-5 polypeptides. Lane C, control with no added template. NS, nonspecific bands. The values on the right are molecular sizes in kilodaltons. (B) Immunofluorescence assays comparing the intracellular localization of HA-tagged IRF-5 V3, V5, and V12 proteins and the phosphomimetic V3 protein (pV3) in transfected HeLa cells. Cells were stained with an anti-HA primary antibody and a rhodamine-labeled secondary antibody. Nuclei were stained with DAPI. (C) Reporter assay in which 293 cells were transfected with an IFNA1 promoter-luciferase reporter in the presence of V3 IRF-5, V12 IRF-5, pV3 IRF-5, or pV3 IRF-5 plus increasing amounts of V12 IRF-5 as indicated. The data shown are averages from two assays.