Transfected cells were amplified and subsequently cloned by serial limit dilution At least 5 clones were screened for each RLR to detect the tagged protein expression by qPCR and Western Blot. For STING37shRLR cell lines, we generated lentiviral vectors using the canonical triple transfection of HEK293T cells by a VSVg envelope, an encapsidation p8, G418 at 500 mg ,
MDA5(RHS4430-101128286-V3LHS_300657, shMDA5) or RIG-I (RHS4430-99619609-V2LHS_197176, shRIG-I), all bearing a puromycin selection marker. Vectors were titrated according to manufacturer's instructions in HeLa cells 2013]) was transduced at an MOI of 0.3 and 48 hr later puromycin (5 mg/ml) was added to the media to select properly transduced cells. CHIKV 06?49 strain was isolated from the serum of an adult patient with arthralgia/ myalgia in Saint Louis city, Ré union CHIKV strain 06? 49 was titrated on VERO cell by TCID50 (50% Tissue Culture Infective Dose) Recombinant CHIKV- Rluc which contains the Rluc reporter gene inserted between the non-structural nsP3 and nsP4 proteins has already been described Attenuated MV Schwarz vaccine strain (MV) and recombinant MV Schwarz expressing Fluc (rMV2-Fluc) from an additional transcription unit derived from MV have been previously described To prevent V protein expression from MV, a two-step PCR strategy was used to generate MVDV virus. Two PCR fragments were amplified using MV2313 (5'-ATCTGCTCCCATCTCTATGG) and MV2504 (5'-TCTGTGCCCTTCTTAATGGG) for the first fragment and MV2485 (5'-CCCATTAA- GAAGGGCACAGA) and MV3385 (5'-AGGTTGTACTAGTTGGGTCG) for the second fragment. These PCRs introduced a mutation interfering with RNA editing (UUAAAAAGGGCACAGA native sequence was mutated to UUAAgAAGGGCACAGA) The two PCR products were combined in a second PCR reaction using MV2313 and MV3385 primers. The produced mutated HindIII-SpeI MV fragment was moved into the pTM-MVSchwarz plasmid after digestion with the corresponding restriction enzymes, Thermo Scientific) expressing either an shRNA with no target (RHS4346, shNeg), or targeting LGP2 (shLGP2) and named MV4V. Immunoblot analysis was performed to validate the lack of MV-V expression by MVDV (data not shown). Virus stocks were produced on VERO cells, and titrated by TCID50, 1997. ,
, Hercules, California) with MOPS running buffer and transferred to cellulose membranes (GE Healthcare, Little Chalfont, United Kingdom) with the Criterion Blotter system (BioRad) The following antibodies were used: an anti-STrEP-Tag (#34850, Qiagen anti-RIG-I (D14G6, Cell Signaling) or monoclonal anti-b-actin antibody (A5441, Sigma), IRF3 (#11904, Cell Signaling) or phosphor-IRF3 (ab76493 HRP-coupled anti-mouse (NA9310V, GE Healthcare) or anti-rabbit (RPN4301, GE Healthcare) were used as secondary antibodies. Peroxidase activity was visualized with an ECL Plus Western Blotting Detection System (#RPN2132, GE Healthcare). MV intracellular staining was performed with mouse anti-N mAb (clone 25 For CHIKV, intracellular staining was performed with either FITC-conjugated anti-CHIK, Antibodies, western blot and intracellular staining Protein extracts were resolved by SDS-polyacrylamide gel electrophoresis on 4?12% Criterion gels (BioRad anti-LGP2 (NBP1- 85348, Novus, Littleton, Colorado), anti-MDA5 (#5321, Cell Signaling, pp.2-1201, 1981.
, 10 5 per well) were plated in a 24-well plate and infected with appropriate MOIs. For rMV2-Fluc or CHIKV-Rluc infection, at each time point cells were lysed by Passive Lysis buffer (E1941, Promega, Fitchburg, Wisconsin), and luciferase activity was measured with Bright- Glow Luciferase assay system (E2650, Promega) or Renilla Glow Luciferase Assay System (E2720, Promega), correspondingly. For immunostaining, cells were washed twice with phosphate-buffered saline (PBS) and 2% foetal calf serum (FCS) and then, MV and CHIKV replication assays STING37shRLR cells
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