Fig. 7: USP18 interacts with TRIM31 to promote the K63-linked polyubiquitination of MAVS.

a Top panel: immunoblot analysis of TRIM31 and actin in HEK293T cells transfected with control siRNA (siControl) or siRNA targeting TRIM31 (siTRIM31) for 12 h followed by transfection of plasmids encoding GFP-TRIM31 for 24 h. Bottom panel: immunoprecipitation analysis of ubiquitination of MAVS in HEK293T cells transfected with siControl and siTRIM31 followed by transfection of the plasmids expressing Flag-MAVS, HA-K63 only Ub, and Myc vector, Myc-USP18. b K63-TUBE analysis of ubiquitination of endogenous MAVS in Trim31^+/+ and Trim31^−/− MEFs transfected with pLenti vector or pLenti-Flag mUSP18 followed by SeV infection for indicated time points. c Left panel, K63-TUBE analysis of ubiquitination of endogenous MAVS in RAW264.7 cells transfected with siControl or siUSP18 followed by infection with SeV for indicated time points. Right panel, K63-TUBE analysis of ubiquitination of endogenous MAVS in Trim31^−/− MEFs transfected with control siRNA or USP18 siRNA followed by SeV infection for indicated time points. d Top panel: schematic diagram of wild-type MAVS and MAVS 3KR mutant (K11, 311, and 461R). Bottom panel: immunoprecipitation analysis of ubiquitination of wild-type-MAVS or MAVS 3KR mutant in HEK293T cells transfected with plasmids expressing Flag-MAVS or Flag-MAVS 3KR, HA-K63 only Ub, and Myc vector or Myc-USP18. e qRT-PCR analysis of IFNB, CCL5, and CXCL10 expression level and immunoblot analysis of MAVS in MAVS KO HeLa cells transfected with control siRNA or USP18 siRNA and reconstituted with GFP, WT MAVS, or MAVS 3KR plasmid followed by infection with SeV for 8 h. Representative data were collected and expressed as mean ± SD from three independent experiments, with **p = 0.0023 (IFNB), ***p < 0.001 (CCL5), ***p < 0.001 (CXCL10) (two-tailed Student’s t test). f Co-IP analysis of the interaction between TRIM31 and USP18 in HEK293T cells transfected with GFP-TRIM31 and Myc-USP18. g Representative confocal images of immunofluorescence staining for Myc-USP18 colocalization with GFP-TRIM31 in HeLa cells, scale bar, 10 µm. Right panel: line profiling of Myc-USP18 with Flag-MAVS, and intensity of each line was quantified by ImageJ software. h Co-IP analysis of the endogenous interaction between TRIM31 and USP18 in THP-1 cells infected with SeV for indicated time points. i Co-IP analysis of interaction between USP18, TRIM31, and MAVS in HEK293T cells transfected with Flag-MAVS, Myc-USP18, and GFP-TRIM31. j Immunoblot analysis of the indicated proteins after isolating the mitochondrial fraction of the MEFs following SeV infection for indicated time points. k Co-IP analysis of interaction between endogenous TRIM31 and MAVS in Usp18^+/+ and Usp18^−/− MEFs infected with SeV for 0–8 h and harvested at indicated times. l Co-IP analysis of interaction between TRIM31 and MAVS in HEK293T cells transfected with HA-MAVS, GFP vector or GFP-TRIM31, and different dosages of Myc-USP18. m In vitro ubiquitination assay of MAVS in the presence or absence of USP18.