Loss of transcriptional function in the IRF8^K108E mutant. (A) RAW macrophages were transiently transfected with increasing amounts of WT (K108) or IRF8^K108E variant (E108) along with a firefly luciferase reporter construct containing the Isg15 promoter and a Renilla luciferase internal control, and luciferase activity was assayed 24 hours later. (B) RAW cells were transiently transfected as in panel A with the addition of IRF1. (C) RAW cells were transiently transfected with expression vectors coding for K108 or with the E108, R108, Q108, and H108 mutants, along with or without IRF1 and an IL-12p40-pGL3 luciferase reporter construct and a Renilla luciferase internal control. Results are represented as means ± standard deviation (SD) of 3 independent experiments. (D) Immunoblots showing similar expression level of K108, and of the E108, R108, Q108, and H108 mutants in transfected cells, and using a mouse anti-HA mAb. Actin expression was evaluated as a loading control.