Fig. 7: RNF115 interacts with and catalyzes K63-linked ubiquitination of MITA after HSV-1 infection. | Nature Communications

Fig. 7: RNF115 interacts with and catalyzes K63-linked ubiquitination of MITA after HSV-1 infection.

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

Fig. 7

a Denature-IP (with control IgG or anti-FLAG) and immunoblot analysis (with anti-HA, FLAG, or RNF115) of HEK293 cells transfected with the indicated plasmids for 24 h. b In vitro ubiquitination analysis of MITA. c Denature-IP (with anti-MITA) and immunoblot analysis (with anti-HA, MITA, or RNF115) of THP-1 cells stably shCon or shRNF115#2 that were transfected with plasmids encoding HA-Ubiquitin or HA-Ubiquitin mutants (K48O, K63O, K48R, K63R) followed by HSV-1 infection for 6 h. d, e Denature-IP (with anti-MITA) and immunoblot analysis (with anti-Ub, K48-linkage polyubiquitin, MITA, RNF115, or β-Tubulin) (d) or SDD-AGE analysis of the aggregation of MITA, native-PAGE analysis of the dimerization of MITA and SDS–PAGE analysis of the indicated proteins (e) in THP-1 cells stably transfected with shCon or shRNF115#2 or in Rnf115+/+ and Rnf115−/− MLFs infected with HSV-1 for 0–6 h. f Immunoprecipitaion (with anti-MITA) and immunoblot analysis (with anti-MITA, TBK1, RNF115, or β-Tubulin) in THP-1 cells stably transfected with shCon and shRNF115#2 (left panels) or in Rnf115+/+ and Rnf115−/− MLFs (right panels) infected with HSV-1 for 0–6 h. g In vitro ubiquitination analysis of ubiquitin-modified MITA or MITA mutants. h Denature-IP (with anti-FLAG) and immunoblot analysis (with anti-FLAG, HA, or GFP) in HEK293 cells transfected with plasmids encoding FLAG-MITA or mutations, HA-Ubiquitin, empty vector and GFP-RNF115 for 24 h. i Denature-IP (with anti-FLAG, upper two panels), immunoprecipitation (with anti-FLAG, middle two panels), and immunoblot analysis (with antiK63 linkage polyubiquitin, FLAG, or TBK1), SDD-AGE analysis (with anti-FLAG or β-Tubulin, middle panels) or native-PAGE analysis (with anti-FLAG) in Mita−/− MLFs reconstituted with MITA or MITA(3KR) followed by infection with HSV-1 for 0–6 h. Data are representative of three (a–c) or two (d–i) independent experiments. Source data are provided as a Source Data file.

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