Materials and Reagents

Materials
1. Autoradiography film (Denville Scientific, catalog number: E3012 )
2. Tissue culture plates (Corning)
3. 1.5 ml Eppendorf tubes (USA Scientific)
4. Centrifuge tubes (Thermo Fisher Scientific)
5. 26-gauge needle
6. PVDF membranes
Cells
1. HT1080 fibrosarcoma cells
  Note: These cells are maintained in DMEM containing 10% FBS, 100 international units of penicillin, 100 µg/ml streptomycin (complete DMEM).
2. 1080.10 cells (IRF3 overexpressing HT1080 cells [Elco et al., 2005])
  Note: These cells are maintained in complete DMEM containing 1 µg/ml puromycin.
3. P2.1/IRF3 cells (P2.1 cells expressing C-terminally Flag-tagged human IRF3, Figure 2A)
Notes:
1. These cells were generated by lentiviral transduction of the P2.1 cells with cDNA of Flag-tagged human IRF3. After lentiviral transduction, the cells were selected in puromycin (1 µg/ml)-containing complete medium. Use the selected pool of cells for the experiments.
2. HT1080 cells were described previously (Chattopadhyay et al., 2016) and the other cell lines (1080.10 and P2.1/IRF3) were generated in the authors’ laboratory.
Viruses
1. Sendai virus (SeV) Cantell strain (Charles River laboratories) – this strain was originally obtained from ATCC (ATCC, catalog number: VR-907^TM )
2. Adenovirus Ad5 strain (a kind gift from Thomas Shenk, Princeton University, New Jersey)
Reagents
1. DMEM (Lerner Research Institute Central Cell Services, catalog number: 11-500p )
2. PolyI:polyC (Poly [I:C]) (GE Healthcare, catalog number: 27-4732-01 )
3. Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific, Invitrogen^TM, catalog number: 11668030 )
4. Opti-MEM I reduced serum medium (Thermo Fisher Scientific, Invitrogen^TM, catalog number: 31985070 )
5. Phosphate-buffered saline (PBS) (Lerner Research Institute Central Cell Services, catalog number: 123-1000p )
6. Recombinant caspase-8 (EMD Millipore, catalog number: CC123 )
7. Protein assay dye (Bio-Rad Laboratories, catalog number: 5000006 )
8. 10x SDS-PAGE running buffer (AMRESCO, catalog number: 0783 )
9. Antibodies:
  anti-Flag (used at 1:2,500) (Sigma-Aldrich, catalog number: F3290 )
  anti-IRF3 (dilution used at 1:10,000) (Active Motif, catalog number: 39033 )
  anti-actin (used at 1:10,000 ) (Sigma-Aldrich, catalog number: A5441 )
  HRP-conjugated secondary antibodies (used at 1:5,000) (Rockland Immunochemicals, catalog numbers: 611-103-121 , 611-103-122 )
10. Fetal bovine serum (FBS) (Atlanta Biologicals, catalog number: S11550 )
11. Proteasome inhibitor MG132 (EMD Millipore, catalog number: 474790 )
12. Phosphatase inhibitor (PhosSTOP) (Roche Diagnostics, catalog number: 04906837001 )
13. cOmplete EDTA-free protease inhibitor (Roche Diagnostics, catalog number: 11873580001 )
14. Caspase inhibitors:
  z-VAD (MBL, catalog number: 4800-520 )
  z-WEHD (MBL, catalog number: BV-1100-3 )
  z-IETD (MBL, catalog number: 4805-510 )
15. Anti-Flag-agarose beads (Sigma-Aldrich, catalog number: A2220 )
16. FLAG peptide (Sigma-Aldrich, catalog number: F3290 )
17. SDS-PAGE loading buffer (Laemelli) (Bio-Rad Laboratories, catalog number: 161-0737 )
18. Tris buffered saline with Tween-20 (TBS-T) (AMRESCO, catalog number: K873 )
19. Nonfat dry milk (Bio-Rad Laboratories, catalog number: 1706404XTU )
20. Pierce^TM ECL2 (Thermo Fisher Scientific, Fisher Scientific, catalog number: PI80196 )
21. Tris buffer (pH 7.4) (Affymetrix, catalog number: 22639 )
22. Sodium chloride (NaCl) (Affymetrix, catalog number: 75888 )
23. Triton X-100 (Sigma-Aldrich, catalog number: T9284 )
24. Sodium orthovanadate (NaVO₄) (Sigma-Aldrich, catalog number: S6508 )
25. Sodium fluoride (NaF) (Sigma-Aldrich, catalog number: S7920 )
26. β-glycerophosphate (Sigma-Aldrich, catalog number: G9422 )
27. Sodium pyrophosphate (Na₂H₂P₂O₇) (Sigma-Aldrich, catalog number: P8135 )
28. HEPES (Sigma-Aldrich, catalog number: H3375 )
29. CHAPS hydrate (Sigma-Aldrich, catalog number: C3023 )
30. EDTA (Affymetrix, catalog number: 15694 )
31. Glycerol (Affymetrix, catalog number: 16374 )
32. 1,4-dithiothreitol (DTT) (Sigma-Aldrich, catalog number: 10197777001 )
33. 10x transfer buffer (AMRESCO, catalog number: 0307 )
34. Cell lysis buffer (see Recipes)
35. Caspase-8 in vitro cleavage assay buffer (see Recipes)

Equipment

Tissue culture incubator (at 37 °C with 5% CO₂) (Thermo Fisher Scientific)
Biosafety cabinet
Table top centrifuge (Eppendorf)
Water bath (Benchmark)
Heating block (at 95 °C) (Benchmark)
SDS-PAGE and transfer apparatus (Bio-Rad Laboratories, model: Mini PROTEAN-II Cell )
Note: This equipment is no longer available at manufacturer.
Rocker (Benchmark)
Rotator (Labnet)
Refrigerator
Autoradiography film processor (Kodak)
Spectrophotometer
Vortex (Thermo Fisher Scientific)

Software

ImageJ (freely available from National Institutes of Health, https://imagej.nih.gov/ij/)

Procedure