Materials and Reagents

1. Huh7 human hepatoma cells, stably replicating a wt JFH1 subgenomic replicon (NS4B^wt) and a recombinant replicon cell line harboring the HA epitope and the adaptive mutation Q31R in NS4B (NS4B^HA) (Paul et al., 2013) and Huh7 hepatoma cells stably overexpressing C-terminally HA-tagged calnexin (CANX^HA) (Paul et al., 2013)
   
2. 100 U/ml Pencillin and Streptomycin (Life Technologies, Gibco^®, catalog number: 15140122 )
   
3. 10% (v/v) Fetal Calf Serum (Life Technologies, Gibco^®, catalog number: 10270-106 )
   
4. 1x nonessential amino acids (Life Technologies, catalog number: 11140-035 )
   
5. Geneticin (Life Technologies, Gibco^®, catalog number: 11811-098 )
   
6. Blasticidin (Life Technologies, Gibco^®, catalog number: R210-01 )
   
7. Bradford reagent
   
8. NaN₃ (Sigma-Aldrich, catalog number: S2002 )
   
9. Hemagglutinin peptide (Sigma-Aldrich, catalog number: I2149 )
   
10. Anti-HA agarose beads (Sigma-Aldrich, catalog number: A2095 )
    
11. 1x protease inhibitor cocktail (Roche Diagnostics, catalog number: 04693116001 )
    
12. Dulbecco’s Modified Eagle’s Medium (DMEM) (Life Technologies, Gibco^®, catalog number: 41965-039 ) (see Recipes)
    
13. Bovine serum albumin (BSA) standard (see Recipes)
    
14. PBS (sterile) (see Recipes)
    
15. Hypotonic buffer (see Recipes)
    
16. Sucrose (see Recipes)
    
17. 5 M NaCl solution (see Recipes)
    
18. IP washing buffer I (see Recipes)
    
19. IP washing buffer II (see Recipes)
    
20. IP elution buffer (see Recipes)
    

Equipment

1. 15 cm cell culture dishes
   
2. 37 °C, 5% CO₂ cell culture incubator
   
3. Cell scraper (e.g. SARSTEDT AG, catalog number: 83.1830 )
   
4. Dounce homogenizer (1 ml) (e.g. WHEATON, catalog number: 357538 )
   
5. Table top cooling centrifuge with swinging buckets for 15 ml and 50 ml falcon tubes (e.g. Heraeus Multifuge 3-SR)
   
6. Open top polyclear ultracentrifuge tubes for SW40 rotor (Seton Identification Products, catalog number: 7031 )
   
7. Ultracentrifuge and SW40 rotor and buckets (e.g. Beckman Optima LE80K Ultracentrifuge and Beckman SW40Ti rotor and buckets)
   
8. Lab stand and clamp
   
9. 19 G needle (e.g. BD, catalog number: 301500 )
   
10. Spectrophotometer (e.g. Amersham Ultrospec 2100 pro) and cuvettes (e.g. SARSTEDT AG, catalog number: 67.742 )
    
11. Rotator wheel for Eppendorf tubes (e.g. Labinco, model: L28, catalog number: 2800 )
    
12. Table top cooling centrifuge for Eppendorf reaction tubes (e.g. Eppendorf, model: 5417R )
    
13. Eppendorf Thermomixer compact
    

Procedure