Arimoto et al. 10.1073/pnas.0611551104. |
Fig. 6. In vitro ubiquitination to RIG-I. Reaction mixtures containing Sepharose 4B beads retaining 0.2 mg of GST-RIG-I, 0.1 mg of UBE1, 0.5 mg of E2 enzymes as indicated and 0.3 mg of ubiquitin in the buffer described in Materials and Methods was incubated at 37°C for 1 h. The GST-RIG-I were eluted from the resins and were subjected to Western blot analysis.
Fig. 7. Nuclear localization of IRF3 was inhibited by RNF125 expression. HeLa cells were transfected with plasmids expressing FLAG-IRF3 or HA-RNF125. Twenty-four hour after transfection, cells were treated with or without Poly I:C. Cells were fixed 12 h after the treatment and analyzed for subcellular localization of IRF3 with an immunofluorescence microscope.
Fig. 8. Ubiquitin conjugation of MDA5 and IPS1 by RNF125. 293FT cells were transfected with plasmids encoding Myc-Ub, either wild-type or C72/75A mutant RNF125 and either FLAG tagged CARD containing region of MDA5 (1-293 aa), FLAG-MDA5CARD (a Left) or FLAG-IPS1 (b Left). Ubiquitination of MDA5CARD and IPS1 were monitored by immunoprecipitation assay. The quantities of MDA5CARD and IPS1 in the immunocomplexes are also shown at the bottom of each panel. Cells were treated with control si-RNA or si-RNF125-3 and were transfected with plasmids expressing Myc-Ub, FLAG-MDA5 (a Right) or FLAG-IPS1 (b Right). Ubiquitin conjugation was monitored by blotting with anti-Myc antibody. Amounts of MDA and IPS1 in cell lysates are shown at the bottom of each panel. All cells were grown in medium containing MG132. (c) In vitro ubiquitination to MDA5 and IPS1. In vitro ubiquitination to MDA5 and IPS1 was essentially the same except by using GST-MDA5 and GST-IPS-1 to that described in the legend to Fig. 6.
Fig. 9. Downstream signal from MDA5 as well as IPS1 was suppressed by RNF125. 293FT cells expressing IFNb-luc, and/or MDA5 (a) and/or IPS1 (b) together with RNF125WT or D76 were analyzed for luciferase activity. 293FT cells treated with control siRNA or siRNA125-3 were transfected with plasmids expressing IFNb-luc and/or MDA5 (a Right) or and/or IPS1 (b Right). Luciferase activity of cell lysates derived from these cells was monitored. (c) As a control experiment, luciferase activity driven by p53 promoter was analyzed obtained from cells expressing p53 and RNF125 ectopically.
SI Materials and Methods
Cell Culture, Transfection, and Luciferase Reporter Assays.
The 293T, HepG2, MEF, and HeLa cells were maintained in DMEM supplemented with 10% FCS, 100 units/ml penicillin, and 100 mg/ml streptomycin in a 10% CO2 humidified atmosphere. Jurkat cells were cultured in RPMI medium 1640 (Nissui) supplemented with 10% FBS, 2 mM L glutamine, and 0.1 mg/ml kanamycin sulfate. Plasmids were transfected by using FuGENE 6 (Roche Applied Science) or lipofectamine (Invitrogen), and siRNA was transfected by siLentFect (BIORAD), according to the manufacturer's instructions. Luciferase activity was normalized to Renilla luciferase activity derived from cotransfected pRL-CMV Luc (Promega). All reporter assays were performed in triplicate. The bars in the figures denote the S.E. The indications in the figures show that the cells were treated for 12 h before lysis with addition of 2 mg or 20 mg/ml poly (I:C) in to culture medium or 4 × 106 or 4 × 105 pfu/ml Sendai virus. Cells were treated with MG132 (final 10 mM) for 12 h before harvest, and cycloheximide was added to culture medium at the final concentration of 50 mg/ml, if found necessary.Yeast Two-Hybrid Screening.
A cDNA encoding human UbcH8 was subcloned into the pGBT9 vector (CLONTECH) in frame with a GAL4 DNA-binding domain to generate bait for the yeast two-hybrid screen. To screen for interacting proteins, we used a human splenic cDNA library as prey. Positive clones were selected by b-gal positivity. We searched DNA databases at the National Center for Biotechnology Information for matches to the cDNA isolated from positive clones with the alignment search tool program BLAST.Western Blotting and Immunoprecipitation.
Samples were denatured in 1× sample buffer [50 mM Tris·HCl (pH 6.8), 2% SDS, 2 mercaptethanol, 10% glycerol, and 1% bromophenol blue] for 5 min at 100°C, and then sonicated for 20 min. To analyze immunocomplexes, cells were either lysed in buffer containing 25 mM Tris·HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, and 0.5% Nonidet P-40 for coimmunoprecipitation assays or in buffer composed of 25 mM Tris·HCl (pH 8.0), 150 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.1% SDS, 1% Nonidet P-40, and 0.5% sodium deoxycholate for immunoprecipitation assays. The soluble fraction was incubated for 1 h with the indicated antibodies. Immunocomplexes were adsorbed to the protein G-Sepharose, and were eluted by boiling for 5 min. Endogeneous immunoprecipitation was performed by using Can get signal (TOYOBO) and Rabbit TrueBlot (eBioscience) immunoprecipitation system according to the manufacturer's instructions. Band intensities were measured with scanning densitometry (Fluor-S MultiImager, Bio-Rad) and depicted as a graph.ELISA.
Culture media was collected and analyzed for IFNb production by using enzyme-linked immunosorbent assays (ELISAs). An ELISA kit for mouse IFNb was purchased from PBL Biomedical Laboratories.In Vitro
Ubiquitin Conjugation Reaction. GST-fused substrates bound to Sepahrose 4B beads were incubated with UBE1(E1), RNF125(E3), ubiquitin, and various E2 enzymes as described in figure legends in 30 ml of 50 mM Tris·HCl (pH 7.5), 10 mM MgCl2, 0.5 mM DTT in the presence of 1 ml of ERS (energy regenerating source; BioMolecular Inc.) at 37°C for 1 h. The beads were washed thoroughly with buffer containing 50 mM Tris·HCl (pH 7.5), 10 mM MgCl2, 0.5 mM DTT, and 1% Triton X-100. The GST-RNF125 and GST-RIG-I were eluted from resin and were subjected to Western blot analysis.Immunofluorescence Analysis.
HeLa cells were seeded on an eight-well chamber slide and transfected with expression plasmids by using FuGENE6. At 36 h, cells were fixed with ice-cold methanol and permeabilized in 0.05% Triton X-100 in PBS at room temperature for 15 min. After blocking with 1% BSA and 10% FBS in PBS for 1 h, cells were incubated with anti-IRF3 (FL425) and anti-HA (12CA5) antibodies at room temperature for 1 h. After washing with PBS four times, samples were incubated with 4,6-diamidino- 2-phenylindole (DAPI) together with Alexa Fluor 488 goat anti-rabbit or 568 goat anti-mouse antibody at room temperature for 30 min. After washing, cells were mounted in immunofluorescence microscope (Leica).