Role of TRIM65 in RNA virus-induced activation of MDA5 signaling. (A and B) qPCR analysis of Ifn-β expression in BMDMs from Mda5^−/− (A) or Trim65^−/− (B) mice infected with indicated MOI of EMCV or VSV for 12 h. (C and D) Secretion of IFN-β (C) or IFN-α (D) by Trim65^−/− BMDMs infected with indicated doses of EMCV or VSV for 12 h. (E) qPCR analysis of Ifn-β expression in BMDMs from WT mice or Trim65^−/− mice lines infected with EMCV or VSV for 12 h. (F) Immunoblot analysis of indicated proteins in Trim65^−/− MEF cells overexpressing GFP-TRIM65 or GFP-TRIM65 without the SPRY domain. (G) qPCR analysis of IFN-β expression in Trim65^−/− MEF cells overexpressing GFP-TRIM65 or GFP-TRIM65 without SPRY domain and left stimulated with EMCV for 12 h. (H and I) qPCR analysis of ISG15 (H) or ISG56 (I) expression in WT or Trim65^−/− BMDMs infected with EMCV (MOI = 0.05) or VSV (MOI = 0.06) for 12 h. (J) Immunoblot analysis of IRF3 phosphorylation (P-IRF3) in WT or Trim65^−/− BMDMs infected with EMCV (MOI = 0.05) for 6 h. (K) qPCR analysis of Ifn-β expression in WT or Trim65^−/− BMDMs infected with adenovirus (AdV; MOI = 1,000) for 12 h. Data are from three independent experiments with biological duplicates in each (A–E, G–I, and K; mean and SEM of n = 6) or representative of three independent experiments (F and J). Statistics were analyzed via an unpaired Student’s t test: **, P < 0.01; ***, P < 0.001.