USP25 is required for RNA virus-triggered signaling in mouse macrophages and dendritic cells. (A) Usp25−/− cells expressed lower levels of Ifna4, Ifnb, Tnf, and/or Il6 mRNA than did the wild-type counterparts after SeV or VSV infection. Wild-type or Usp25−/− BMDMs, BMDCs, FLT3L-pDCs were infected with SeV or VSV for the indicated time points before qPCR analysis was performed. (B) Usp25−/− cells produced reduced IFN-α and IL-6 proteins than did the wild-type counterparts after SeV or VSV infection. Wild-type or Usp25−/− BMDMs, BMDCs, FLT3L-pDCs were infected with SeV or VSV for 12 h and the supernatants were collected for ELISA analysis. (C) SeV-induced phosphorylation of IRF3 and IκBα was impaired in Usp25−/− BMDCs compared with wild-type BMDCs. Wild-type or Usp25−/− BMDCs were infected with VSV for 6–12 h. Cells were lysed and the lysates were subject to immunoblot analysis with the indicated antibodies. Data are representative of three independent experiments (mean and SD of three replicates in A and B).