Fig. 6: RNF115 mediates K48-linked ubiquitination and proteasomal degradation of homeostatic MAVS. | Nature Communications

Fig. 6: RNF115 mediates K48-linked ubiquitination and proteasomal degradation of homeostatic MAVS.

From: RNF115 plays dual roles in innate antiviral responses by catalyzing distinct ubiquitination of MAVS and MITA

Fig. 6

a Denature-immunoprecipitation (Denature-IP) (with anti-FLAG) and immunoblot analysis (with anti-FLAG, anti-HA, or anti-GFP) of HEK293 cells transfected with plasmids encoding FLAG-MAVS, HA-Ubiquitin, and empty vector, GFP-RNF115, or GFP-RNF115(2CA) for 24 h. b In vitro ubiquitination analysis of ubiquitin-modified MAVS. MAVS, RNF115, and RNF115(2CA) were translated in vitro, Biotin-Ub, E1, and UbcH5 were added for ubiquitination assays. Ubiquitin-conjugated MAVS was detected by immunoblot with HRP–streptavidin (upper panel). The proteins in the input were detected by immunoblot with the indicated antibodies (lower panels). c Immunoblot of HeLa cells that were transfected with plasmids encoding RNF115 (0, 200, 400, 600 ng, respectively) and RNF115(2CA) (400 ng) for 24 h (upper left panels), or transfected with shRNA(Con, shRNF115#1 or shRNF115#2) for 36 h (upper right panels), lysed and analyzed by immunoblot with anti-MAVS, RNF115, and β-Tubulin. Immunoblot analysis of RNF115, MAVS, and GAPDH in brains, kidneys, livers from Rnf115+/+ and Rnf115−/− mice, or in Rnf115+/+, Rnf115+/−, and Rnf115−/− MEFs (lower panels). d Immunoblot analysis of MAVS, RNF115, and GAPDH in HeLa cells that were transfected with shCon or shRNF115#2 for 36 h, in Rnf115+/+ and Rnf115−/− MLFs treated with MG132 for 0–8 h or in human CD14+ monocytes-derived macrophages transfected with control siRNA (NC) or siRNF115 followed by treatment of MG132 for 0–6 h. e Denature-IP (with anti-MAVS) and immunoblot analysis (with anti-K48 linkage polyubiquitin, anti-MAVS, anti-RNF115, or anti-GAPDH) in HeLa cells that were transfected with shCon and shRNF115#2 for 36 h or in Rnf115+/+ and Rnf115−/− MLFs treated by MG132 for 0–6 h. f Denature-IP (with anti-FLAG) and immunoblot analysis (with anti-K48 linkage polyubiquitin, anti-FLAG, or anti-GAPDH) of Mavs−/− MLFs reconstituted with MAVS or MAVS(K500R) treated with MG132 for 0–6 h. Data are representative of three (a, b) or two (c–f) independent experiments. Source data are provided as a Source Data file.

Back to article page