Fig. 5: USP18 promotes the K63-linked polyubiquitination and subsequent aggregation of MAVS. | Nature Communications

Fig. 5: USP18 promotes the K63-linked polyubiquitination and subsequent aggregation of MAVS.

From: USP18 positively regulates innate antiviral immunity by promoting K63-linked polyubiquitination of MAVS

Fig. 5

a Immunoprecipitation analysis of ubiquitination of signaling molecules in HEK293T cells transfected with the plasmids expressing Myc-MAVS or STING, HA-Ub, and Flag vector or Flag-USP18. b Immunoprecipitation analysis of ubiquitination of MAVS in HEK293T cells transfected with the plasmids expressing Flag-MAVS, HA-wild-type, K63 only, or K48 only Ub, and Myc vector or Myc-USP18. c Immunoblot analysis of ubiquitination of endogenous MAVS in RAW264.7 cells transfected with siControl or siUSP18 followed by infection with SeV for indicated time points. d K63-Ub-TUBE analysis of ubiquitination of endogenous MAVS in Usp18+/+ and Usp18−/− MEFs infected with SeV and harvested at indicated time points. e SDD-AGE (top) and SDS-PAGE (bottom) immunoblot analysis of MAVS aggregation in HEK293T cells transfected with the plasmids expressing Flag-MAVS with Myc vector or Myc-USP18. f Representative confocal images of immunofluorescence staining for HeLa cells transfected with the plasmids expressing Flag-MAVS, DsRED2-Mito together with Myc vector or Myc-USP18, scale bar, 10 µm. g Quantification of MAVS aggregate in HeLa cells transfected with the plasmids expressing Flag-MAVS, DsRED2-Mito together with Myc vector or Myc-USP18 (50 cells per sample). Data are presented with mean ± SD, with **p = 0.0019 (two-tailed Student’s t test). h SDD-AGE (top) and SDS-PAGE (bottom) immunoblot analysis of MAVS aggregation in Usp18+/+ and Usp18−/− MEFs infected with SeV and harvested at indicated time points. i Representative confocal images of immunofluorescence staining for Usp18+/+ and Usp18−/− MEFs harvested before infection or infection with SeV for 8 h, scale bar, 10 µm. j Quantification of MAVS aggregate in Usp18+/+ and Usp18−/− MEFs harvested before infection or infection with SeV for 8 h (30 cells per sample). Data are presented with mean ± SD, with *p = 0.0328 (two-tailed Student’s t test).

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