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. 2017 Nov 20;13(11):e1006693. doi: 10.1371/journal.ppat.1006693

Fig 5. iRhom2 antagonizes RNF5-mediated degradation of VISA.

Fig 5

(A) Immunoblot analysis of the indicated proteins in HEK293 cells that were transfected with iRhom2-shRNA plasmid and selected with puromycin (1 μg/ml) for 2 days before infected with SeV for the indicated times. (B) Immunoblot analysis of the indicated proteins in iRhom2+/+ and iRhom2–/–BMDCs infected with SeV for the indicated times. (C) Detection of the association of iRhom2 with several E3 ligases in HEK293T cells transfected with the indicated plasmids by co-immunoprecipitation and immunoblot analysis with the indicated antibodies. (D) Detection of the association of iRhom2 with RNF5 by endogenous co-immunoprecipitation and immunoblot analysis with the indicated antibodies in HEK293 cells un-infected or infected with HSV-1 for the indicated times. (E) Detection of effects of iRhom2 on the expression of AIP4, Sumrf1, VCP, PSMA7 and RNF5 in HEK293T transfected with the indicated plasmids by co-immunoprecipitation and immunoblot analysis were performed with the indicated antibodies. (F) Immunoblot analysis of the indicated proteins in HEK293T cells that were transfected with iRhom2-shRNA plasmid and selected with puromycin (1 μg/ml) for 2 days before infected with SeV for the indicated times. (G) qPCR analysis of RNF5 mRNA in HEK293T cells that were transfected with iRhom2-RNAi plasmid and selected with puromycin (1 μg/ml) for 36 h before infected with SeV for the indicated times. (H) Immunoblot analysis of the indicated proteins in HEK293 cells that were transfected with iRhom2-shRNA plasmid and selected with puromycin (1 μg/ml) for 2 days before treated with cycloheximide (CHX; 100 μg/ml) for the indicated times. (I) Immunoblot analysis of the indicated proteins in wild-type and RNF5-KO HEK293T cells transfected with iRhom2-shRNA plasmid for 2 days before infected with SeV for the indicated times. (J) qPCR analysis of IFNB1 and IRHOM2 mRNAs in wild-type and RNF5-KO HEK293T cells transfected with iRhom2-shRNA plasmid for 2 days before infected with SeV for 5 h. *P < 0.05; **P < 0.01 (unpaired t-test). Data are representative of three experiments with similar results.