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. 2017 Apr 3;214(4):973–989. doi: 10.1084/jem.20161015

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© 2017 Hu et al.

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Figure 8. — Sumoylation of the CARDs of MDA5 and RIG-I is critical for their recruitment of PP1α. (A) Effects of MDA5, RIG-I, and their mutants on activation of the IFN-β promoter. HEK293 cells were transfected with the indicated plasmids for 24 h before luciferase assays. (B) Effects of sumoylation-defective mutation of the CARDs of RIG-I and MDA5 on their dephosphorylation, K63-linked polyubiquitination and recruitment of PP1α after viral infection. The reconstituted MEFs were left uninfected or infected with SeV or EMCV for the indicated times, followed by immunoprecipitation. The immunoprecipitates were divided into two equal portions, and one was used for immunoblot analysis with the PP1α antibody and the other was lysed in denaturing conditions and reimmunoprecipitated for endogenous ubiquitination detection. (C) Effects of sumoylation of RIG-I-CARD or MDA5-CARD on their interactions with PP1α. HEK293 cells were transfected with the indicated plasmids for 24 h, followed by coimmunoprecipitation and immunoblotting analysis. (D) Effects of RIG-I-, MDA5- and their mutants on activation of the IFN-β promoter. HEK293 cells were transfected with the indicated plasmids for 24 h followed by luciferase assays. Data in A and d are from one representative experiment with three technical replicates (mean ± SD). All the experiments were repeated three times.