PolyI:C was purchased from GE Healthcare company, and solved in milliQ water. For polyI:C treatment, polyI:C (50 μg/mL)
was mixed with DEAE-dextran (0.5 mg/mL) (Sigma) in the culture medium, and the cell culture supernatant was replaced with the medium containing polyI:C and DEAE-dextran. Using DEAE-dextran, polyI:C is incorporated into the cytoplasm to activate RIG-I/MDA5. VSV Indiana strain or poliovirus type 1 Mahoney strain were used for virus assay. this website Vero derived cell (Vero-SLAM) was used for propagation and plaque assay for VSV indiana strain or poliovirus type 1 Mahoney strain. HEK293 cells were infected with viruses at MOI=0.001 in a 24-well plate. The virus titers of culture media at indicated hours post infection in the figures were determined by plaque assay using Vero-SLAM cells. In some experiments that require rapid virus propagation, high MOI (0.1∼1) was used for infection. HEK293FT cells were transfected in a 6-well plate with plasmids encoding DDX3, IPS-1, RIG-I or MDA5 as indicated in the figures. Twenty-four hours after tranfection, the total cell lysate
was prepared by lysis buffer (20 mM Tris-HCl (pH 7.5) containing 125 mM NaCl, 1 mM EDTA, 10% glycerol, 1% NP-40, 30 mM NaF, 5 mM Na3Vo4, 20 mM IAA and 2 mM PMSF), and the protein was immunoprecipitated Selleckchem MK-2206 with anti-HA polyclonal (Sigma) or anti-FLAG M2 mAb (Sigma). The precipitated samples were resolved on SDS-PAGE, blotted onto a nitrocellulose sheet and stained with anti-HA (HA1.1) monoclonal (Sigma), anti-HA polyclonal or anti-FLAG M2 mAb. HeLa cells were plated onto cover glass in a 24-well plate. In the CYTH4 following day, cells were transfected with indicated plasmids using Fugene HD (Roch). The amount of DNA was kept constant by adding empty vector. After 24 h, cells were fixed with 3% of paraformaldehyde in PBS for 30 min, and then permeabilized with PBS containing 0.2% of Triton X-100 for 15 min. For the polyI:C stimulation, 100 ng of polyI:C were transfected into HeLa cell in 24-well plates together with IPS-1 or DDX3 expressing vectors, and 24 h after
the transfection, the cells were fixed and stained for confocal microscopic analysis. Permeabilized cells were blocked with PBS containing 1% BSA and were labeled with anti-Flag M2 mAb (Sigma), anti-HA polyclonal Ab (Sigma) or Mitotracker in 1% BSA/PBS for 1 h at room temperature. The cells were then washed with 1% BSA/PBS and treated for 30 min at room temperature with Alexa-conjugated Ab (Molecular Probes). Thereafter, micro-cover glass was mounted onto slide glass using PBS containing 2.3% DABCO and 50% of glycerol. The stained cells were visualized at ×60 magnification under a FLUOVIEW (Olympus, Tokyo, Japan). The authors thank Dr. M. Sasai, Dr. T. Ebihara, Dr. K. Funami, Dr. A. Matsuo, Dr. A. Ishii, Dr. A. Watanabe and Dr. M. Shingai in our laboratory for their critical discussions.