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. 2015 Aug 24;112(36):11324–11329. doi: 10.1073/pnas.1509968112

Fig. S7.

Fig. S7.

USP25 interacts with TRAF3 and TRAF6 through the TRAF-interacting motifs (TIMs). (A) An alignment of the conserved TIMs of USP25. (B) Mutation of the TIMs of USP25 impaired TRAF-USP25 interaction. HEK293 cells were transfected with HA-TRAF3 or HA-TRAF6 (3 μg) and Flag-tagged USP25, USP25(ΔT1), USP25(ΔT2) or USP25(ΔT1+2) (5 μg). Twenty hours later, coimmunoprecipitation and immunoblot analysis was performed with the indicated antibodies. (C) Reconstitution of USP25(ΔT1+2) into Usp25−/− MEFs failed to fully restore the expression of Ifnb and Tnf after SeV or HSV-1 infection. Usp25−/− MEFs were reconstituted with an RVKM-IRES-GFP empty vector (Vec), RVKM-USP25, or RVKM-USP25(ΔT1+2) through retrovirus-mediated gene transfer. Forty hours later, the expression of transduced proteins was examined by immunoblot analysis (Left) and cells were infected with SeV or HSV-1 for 12 h followed by qPCR analysis (Right). Data are representative of two independent experiments (mean and SD of three replicates in C). *P < 0.05, **P < 0.01, ***P < 0.001.