Supplementary Figure 1

Transcription

1 Supplementary Figure 1 Virus infection induces RNF128 expression. (a,b) RT-PCR analysis of Rnf128 (RNF128) mrna expression in mouse peritoneal macrophages (a) and THP-1 cells (b) upon stimulation with poly(i:c) and ISD or infection with SeV and HSV-1 for indicated times. Data are representative of three independent experiments with similar results.

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4 Supplementary Figure 2 RNF128 positively regulates IFN-β signaling. (a) Quantitative RT-PCR analysis of Rnf128 mrna expression (upper) and western blot analysis of RNF128 protein expression (lower) in mouse peritoneal macrophages transfected with control sirna or mice RNF128 sirna 1, 2 or 3 for 48 h. (b) mrna expression of Ifnb1, Cxcl10, Mx1, Ccl5, Il6 and Tnfa in mouse peritoneal macrophages transfected with control sirna or mice RNF128 sirna 1, 2 or 3 and then stimulated with poly(i:c) and ISD or infected with SeV and HSV-1. (c) Quantitative RT-PCR analysis of RNF128 mrna expression (upper) and western blot analysis of RNF128 protein expression (lower) in THP-1 cells transfected with control sirna or human RNF128 sirna 1, 2 or 3 for 48 h. (d) Quantitative RT-PCR analysis of IFNB1, CXCL10, MX1, CCL5, IL6 and TNFA mrna expression in THP-1 cells transfected with control sirna or human RNF128 sirna 1, 2 or 3 for 48 h and then stimulated with poly(i:c) and ISD or infected with SeV and HSV-1. (e) Quantitative RT-PCR analysis of Ifnb1, Cxcl10, Mx1, Ccl5, Il6 and Tnfa mrna expression in WT or Rnf128 / mouse peritoneal macrophages stimulated with LPS for the indicated time. (f,g) Luciferase activity of IFN-β promoter reporter and mrna expression of IFNB1 in HEK293 cells transfected with expression plasmids for TRIF, cgas+sting and RIG-IN along with V5-RNF128 expression plasmid or control vector for 24 h. *p<0.05, # p<0.01 (two-tail Student's t-test). Data are from three independent experiments (a-f; mean+s.d. of triplicate assays) or are representative of three independent experiments with similar results (a,b).

5 Supplementary Figure 3 RNF128 promotes IRF3 activation. (a) Luciferase activity in HEK293 cells transfected with IFN-β, PRDⅠ-Ⅲ, PRDⅡ, PRDⅣ luciferase reporter and RNF128 expression plasmid together with expression plasmids for TRIF, MAVS, cgas+sting or control plasmid. (b) Western blot analysis of phosphorylated-irf3 in HEK293 cells transfected with RNF128 expression plasmid or control plasmid, along with expression plasmids for TRIF, MAVS, cgas+sting or control plasmid. (c) Western blot analysis of phosphorylated-irf3 protein in HEK293 cells transfected with expression plasmids for MAVS and increased amount of V5-RNF128 plasmid. *p<0.05,** p<0.01 (two-tail Student's t-test). Data are from three independent experiments (a; mean+s.d. of triplicate assays) or are representative of three independent experiments with similar results (b,c).

6 Supplementary Figure 4 RNF128 targets TBK1. (a) Coimmunoprecipitation analysis of RNF128 interaction with TBK1 in THP-1 cells infected with SeV or HSV-1 for 8 h. (b) ISRE luciferase activity in HEK293 cells transfected with V5-RNF128 expression plasmid or mutant plasmids together with expression plasmids as indicated. (c) Western blot analysis of phosphorylated-irf3 protein in HEK293 cells transfected with expression plasmids for RNF128 expression plasmid or the mutants and MAVS or cgas and STING expression plasmids. (d) Coimmunoprecipitation analysis of MAVS ubiquitination in primary macrophages from WT and Rnf128 / mice were infected with SeV. (e) Coimmunoprecipitation analysis of STING ubiquitination in primary macrophages from WT and Rnf128 / mice were infected with HSV-1. **p<0.01 (two-tail Student's t-test). Data are representative of three independent experiments with similar results (a,c-e) or are from three independent experiments (b; mean+s.d. of triplicate assays).

7 Supplementary Figure 5 RNF128 positively regulates TBK1 kinase activity. (a) TBK1kinase activity assays in HEK293 cells transfected with the RNF128 expression plasmid and then infected with SeV for 6 h. (b) TBK1 kinase activity assays in peritoneal macrophages transfected with mice RNF128 sirna, then the cells were infected with SeV for 6 h. (c) Quantitative RT-PCR analysis of mrna expression of Ifnb1 (upper) and western blot analysis of phosphorylated-tbk1 protein (lower) in TBK1 deficient MEFs transfected with expression plasmids for Myc-TBK1-WT or Myc-TBK1-S172A along with V5-RNF128 plasmids. *p<0.05, **p<0.01, ns, not significant (twotail Student's t-test). Data are from three independent experiments (a,b; mean+s.d. of triplicate assays) or are representative of three independent experiments with similar results (c-h).

8 Supplementary Figure 6 RNF128 positively regulates antiviral response. (a) VSV titers and VSV-G protein in HEK293 ( ) transfected with the V5-RNF128-WT, mutant V5-RNF128-H2N2 and V5-RNF128- PA expression plasmid and then infected with VSV (MOI, 0.1). (b) VSV titers and VSV-G protein in mouse peritoneal macrophages transfected with control sirna or RNF128 sirna and then infected with VSV (MOI, 0.1). (c) HSV-1 genomic DNA and titers in mouse primary macrophages transfected with control sirna or RNF128 sirna and then infected by HSV-1 (MOI, 10). (d) VSV titers and VSV-G protein in THP-1 cells transfected with control sirna or RNF128 sirna and then infected with VSV (MOI, 0.1). (e) HSV-1 titers and genomic DNA in THP-1 cells transfected with control sirna or RNF128 sirna and then infected by HSV-1 (MOI, 10). *p<0.05, **p<0.01 (two-tail Student's t-test). Data are from three independent experiments (a-e; mean+s.d. of triplicate assays) or are representative of three independent experiments with similar results (a,b,d).

9 Supplementary Table 1 Antibody Clone Distributor Anti- HA sc-7392 Santa Cruz Biotechnology Anti- Ub sc-8017 Santa Cruz Biotechnology Anti- STAT1 sc-417 Santa Cruz Biotechnology Anti- GAPDH sc Santa Cruz Biotechnology anti- RNF128 H BD Biosciences Anti- V5 ab53418 Abcam Anti- HSV-1 ICP8 ab20194 Abcam Anti-Myc #2276 Cell Signaling Technology Anti-IRF3 #4302 Cell Signaling Technology Anti-TBK1 #3504 Cell Signaling Technology Anti-phospho-IRF3 #29047 Cell Signaling Technology Anti-phospho-TBK1 #5483 Cell Signaling Technology Anti-PCNA #13110 Cell Signaling Technology Anti-STAT2 #72604 Cell Signaling Technology Anti-ubiquitin K63 #5621 Cell Signaling Technology Anti-ubiquitin K48 #8081 Cell Signaling Technology Anti-Flag F1804 Sigma-Aldrich Anti-VSV-G V5507 Sigma-Aldrich Anti-IFNAR1 MAR1-5A3 Leinco Technologies Supplementary Table 2 Gene Species Sense primer Antisense primer RNF128 Human 1 GAGGCAAUUAAAGGCAGAUTT AUCUGCCUUUAAUUGCCUCTT Human 2 GGCAGUGGAUGUUAUUCCUTT AGGAAUAACAUCCACUGCCTT Human 3 GUCCCUGUAUCCAAUGAAATT UUUCAUUGGAUACAGGGACTT RNF128 Mouse 1 GGUCAUCGAAGUAGGGAAATT UUUCCCUACUUCGAUGACCTT Mouse 2 GGGUGAAUCAUUAUUCAAUTT AUUGAAUAAUGAUUCACCCTT Mouse 3 GGUAAACCAUGAAGCAAAUTT AUUUGCUUCAUGGUUUACCTT Mib1 Mouse 1 GGUUCUGUUAGAGUCUCGUTT ACGAGACUCUAACAGAACCTT Mouse 2 CUAGUCAGAACGGACAUGUTT ACAUGUCCGUUCUGACUAGTT Mouse 3 CGGAGUCCUUCUAUGAUUATT UAAUCAUAGAAGGACUCCGTT Mib2 Mouse 1 CUCUAUGACAACGCCCAAATT UUUGGGCGUUGUCAUAGAGTT Mouse 2 CGCUAUGAGACAUCUCACUTT AGUGAGAUGUCUCAUAGCGTT Mouse 3 CCUAAGGGAUAUGCAAGAATT UUCUUGCAUAUCCCUUAGGTT Nrdp1 Mouse 1 CCUAUUUGCAGUGGAGUCUTT AGACUCCACUGCAAAUAGGTT Mouse 2 GAGGAGACAAUCGAAUACATT UGUAUUCGAUUGUCUCCUCTT Mouse 3 GGCGCUACUAUGAGAACUATT UAGUUCUCAUAGUAGCGCCTT Scramble Human Mouse UUCUCCGAACGUGUCACFUTT UUCUCCGAACGUGUCACFUTT ACGUGACACGUUCGGAGAATT ACGUGACACGUUCGGAGAATT

10 Supplementary Table 3 Gene Species Forward primer (5'-3') Reverse primer (5'-3') Rnf128 Mouse AGCTTCCATAATAAACACCT CCTTTAACTGCCTCTGTAATAA RNF128 Human TGGTTTCATCCATTCAGCAA AGTTTGAAGCTTCATCCACCA Ifnb1 Mouse AGTTACACTGCCTTTGCC GTTGAGGACATCTCCCAC IFNB1 Human CAACAAGTGTCTCCTCCAAAT TCTCCTCAGGGATGTCAAAG Il6 Mouse GCCTTCTTGGGACTGATGCT CTGCAAGTGCATCATCGTTGT IL6 Human ACCCCCAATAAATATAGGACTGGA TCTTCTCCTGGGGGTACTGG Tnfa Mouse AGGCACTCCCCCAAAAGATG CCACTTGGTGGTTTGTGAGTG TNFA Human CACAGTGAAGTGCTGGCAAC AGGAAGGCCTAAGGTCCACT Cxcl10 Mouse CCAAGTGCTGCCGTCATTTT GATAGGCTCGCAGGGATGAT CXCL10 Human TGCCATTCTGATTTGCTGCC TGATGGCCTTCGATTCTGGA Mx1 Mouse CATCAGAGTGCAAGCGAGGA TAAACAACCGGCTGTCTCCC MX1 Human CTCCGACACGAGTTCCACAA GGCTCTTCCAGTGCCTTGAT Ccl5 Mouse CCTCACCATATGGCTCGGAC TCTTCTCTGGGTTGGCACAC CCL5 Human CCCCATATTCCTCGGACACC GCTGTCCCTCTCTCTTTGGC Gapdh Mouse GGAGAGTGTTTCCTCGTCCC ACTGTGCCGTTGAATTTGCC GAPDH Human AATGGGCAGCCGTTAGGAAA GCGCCCAATACGACCAAATC