Materials and Reagents

1. 6 well dish (Corning, Falcon^®, catalog number: 353046 ) (Stored at room temperature)
   
2. PVDF membrane (GE Healthcare, catalog number: 10600023 ) (Stored at room temperature)
   
3. DT40 cell line (DT40 is a B cell line derived from an avian leukosis virus induced bursal lymphoma in a white leghorn chicken) (ATCC, catalog number: CRL-2111 ) (Endogenous expression of cytosolic PNGase)
   
4. Homo sapiens colon colorectal carcinoma cell line (HCT116) (ATCC, catalog number: CCL-247 ), HCT116 cells (ATCC, catalog number: CCL-247) (Endogenous expression of cytosolic PNGase)
   
5. Opti-mem (Thermo Fisher Scientific, Gibco^TM, catalog number: 31985-070 )
   
6. Lipofectamine 2000 (Thermo Fisher Scientific, Invitrogen^TM, catalog number: 11668019 )
   
7. Dulbecco’s modified Eagle’s medium (DMEM) (NACALAI TESQUE, catalog number: 08458-45 ) (Stored at 4 °C)
   
8. Fetal bovine serum (FBS) (Thermo Fisher Scientific, Gibco^TM, catalog number: 10270-106 )
   
9. 100 U/ml penicillin and 100 μg/ml streptomycin (NACALAI TESQUE) (Stored at -20 °C)
   
10. RPMI (NACALAI TESQUE, catalog number: 30263-95 ) (Stored at 4 °C)
    
11. Chicken serum (Thermo Fisher Scientific, Gibco^TM, catalog number: 16110-082 )
    
12. 1 M dithiothreitol (DTT) (Wako Pure Chemical Industries, catalog number: 041-08976 ) (for reduction of proteins; in water; stored at -20 °C)
    
13. 20 mM carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-fmk) (Promega, catalog number: G7231 ) (for inhibition of PNGase activity; stored at -20 °C)
    
14. Nonidet P-40 (NACALAI TESQUE, catalog number: 23640-94 ) (for HCT116; stored at room temperature)
    
15. 4% digitonin (Wako Pure Chemical Industries, catalog number: 043-21371 ) (for DT40; in water; stored at -80 °C)
    
16. Anti-myc antibody (MEDICAL & BIOLOGICAL LABORATORIES, catalog number: 562 ) (Stored at -20 °C)
    
17. Anti-β-actin antibody conjugated with HRP (Wako Pure Chemical Industries, catalog number: 017-24573 )
    
18. NaCl (Wako Pure Chemical Industries, catalog number: 191-01665 )
    
19. Na₂HPO₄
    
20. KCl
    
21. KH₂PO₄
    
22. Tris/HCl, pH 8.0 (Sigma-Aldrich, catalog number: T6791 ) (Stored at room temperature)
    
23. Glycerol
    
24. Bromophenol blue (BPB)
    
25. Sodium dodecyl sulfate
    
26. Protease inhibitor cocktail (100x) (NACALAI TESQUE, catalog number: 25955-11 ) (for inhibition of various proteases’ activity; in ddH₂O; stored at -20 °C)
    
27. 10 mM Z-Leu-Leu-Leu-CHO (MG132) (PEPTIDE INSTITUTE, catalog number: 3175-v ) (for inhibition of proteasomal activity; in DMSO; stored at -20 °C)
    
28. Phosphate buffered saline (PBS) (see Recipes)
    
29. 2x sodium dodecyl sulfate (SDS) sample buffer (pH 6.8) (see Recipes)
    
30. Buffer A (Stored at 4 °C) (see Recipes)
    
31. Buffer B (see Recipes)
    

Equipment

1. High speed refrigerated micro centrifuge (Tomy, model: MX-301 )
   
2. mPAGE (ATTO, model: AE-6530 ) (Using hand-made 10% gel)
   
3. Transfer equipment (ATTO, model: WSE-4020 )
   
4. Heat block (TAITEC, model: DTU-1BN )
   
5. Micro porator (Digital Bio, model: MP-100 )
   

Software

1. ImageJ

Procedure