Fig. 4: USP18 targets MAVS in the RLR signaling pathway.

a Co-IP analysis of the interaction between USP18 and signaling molecules in HEK293T cells transfected with the plasmids expressing Flag-USP18 and Myc-RIG-I, MDA5, MAVS, TBK1, and IRF3. Asterisk represents unspecific signal. b Top panel: schematic representation of MAVS or MAVS truncations. Bottom panel: Co-IP analysis of the interaction between Flag-USP18 and Myc-MAVS or MAVS truncations in HEK293T cells. c Top panel: representative confocal images of immunofluorescence staining for Myc-USP18 only or Myc-USP18 colocalization with Flag-MAVS in HeLa cells, scale bar, 10 µm. Bottom panel: line profiling of Myc-USP18 with Flag-MAVS, and intensity of each line was quantified by ImageJ software and drawn by GraphPad Prism 8.0. d In situ PLA assay of the USP18-MAVS interaction in HeLa cells with indicated combinations using an anti-Flag and anti-Myc antibodies, scale bar, 10 µm. e Co-IP analysis of the interaction between USP18 and MAVS in THP-1 cells infected with SeV for 0–8 h, lysed and immunoprecipitated with a control immunoglobulin G (IgG), or anti-MAVS. f Left panel: In situ PLA assay of the USP18-MAVS interaction in THP-1 cells infected without or with SeV using anti-USP18 and anti-MAVS antibodies, scale bar, 10 µm. Right panel: quantification of interaction spots before and after SeV infection from 50 cells was drawn by GraphPad Prism 8.0. g Co-IP analysis of the interaction between USP18 and MAVS in RAW264.7 cells infected with SeV for 0–8 h. h Co-IP analysis of the interaction between USP18 and MAVS in the mitochondrial (Mito) fraction of THP-1 cells infected with SeV for 0–8 h.