Materials and Reagents

1. 6 well dish (Corning, Falcon^®, catalog number: 353046 )
   
2. PVDF membrane (GE Healthcare, catalog number: 10600023 )
   
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 )
   
4. Homo sapiens colon colorectal carcinoma cell line (HCT116) (ATCC, catalog number: CCL-247 )
   
5. Dulbecco’s modified Eagle’s medium (DMEM) (NACALAI TESQUE, catalog number: 08458-45 )
   
6. Fetal bovine serum (FBS) (Thermo Fisher Scientific, Gibco^TM, catalog number: 10270-106 )
   
7. 100 U/ml penicillin and 100 μg/ml streptomycin (NACALAI TESQUE)
   
8. RPMI (NACALAI TESQUE, catalog number: 30263-95 )
   
9. Chicken serum (Thermo Fisher Scientific, Gibco^TM, catalog number: 16110-082 )
   
10. Opti-mem (Thermo Fisher Scientific, Gibco^TM, catalog number: 31985-070 )
    
11. Lipofectamine 2000 (Thermo Fisher Scientific, Invitrogen^TM, catalog number: 11668019 )
    
12. 2.5 g/L trypsin (NACALAI TESQUE, catalog number: 32777-44 )
    
13. Protease inhibitor cocktail (100x) (NACALAI TESQUE, catalog number: 25955-11 ) (for inhibition of various proteases’ activity such as trypsin, in ddH₂O; stored at -20 °C)
    
14. NaCl (Wako Pure Chemical Industries, catalog number: 191-01665) (for DT40)
    
15. Na₂HPO₄
    
16. KCl
    
17. KH₂PO₄
    
18. Tris/HCl (pH 8.0) (Sigma-Aldrich, catalog number: T6791 ) (Stored at room temperature)
    
19. Glycerol
    
20. Bromophenol blue (BPB)
    
21. Sodium dodecyl sulfate
    
22. Nonidet P-40 (NACALAI TESQUE, catalog number: 23640-94 ) (for HCT116)
    
23. 20 mM carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-fmk) (Promega, catalog number: G7231 ) (for inhibition of PNGase activity)
    
24. 10 mM Z-Leu-Leu-Leu-CHO (MG132) (PEPTIDE INSTITUTE, catalog number: 3175-v ) (in DMSO; inhibition of proteasomal activity, stored at -20 °C)
    
25. 1 M dithiothreitol (DTT) (Wako Pure Chemical Industries, catalog number: 041-08976 ) (in water; for reduction of proteins)
    
26. Anti-β-actin antibody (Wako Pure Chemical Industries, catalog number: 017-24573 )
    
27. Anti-myc antibody (MEDICAL & BIOLOGICAL LABORATORIES, catalog number: 562 )
    
28. Phosphate buffered saline (PBS) (see Recipes)
    
29. 2x sodium dodecyl sulfate (SDS) sample buffer (pH 6.8) (see Recipes)
    
30. Buffer A (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