Detection of ASC Oligomerization by Western Blotting

Materials and Reagents

1. 6 well culture dishes (TPP, catalog number: 92406 )
   
2. Syringe 1 ml (BD, catalog number: 300013 )
   
3. 21 gauge needle (B. Braun Melsungen, Sterican^®, catalog number: 4657527 )
   
4. 1.5 ml Eppendorf tubes (Corning, Axygen^®, catalog number: MCT-150-C-S )
   
5. 200 μl pipette tips (STARLAB INTERNATIONAL, TipOne, catalog number: S1111-1000 )
   
6. 1,000 μl pipette tips (STARLAB INTERNATIONAL, TipOne, catalog number: S1111-6001 )
   
7. Cell scrapers (Corning, Falcon^®, catalog number: 352340 )
   
8. Nitrocellulose blotting membrane (Amersham) (GE Healthcare, catalog number: 10600003 )
   
9. Immortalized Murine Bone-Marrow-Derived Macrophages (iBMDMs), obtained from Professor Petr Broz, Biozentrum, University of Basel, Switzerland
   
10. Opti-MEM (Thermo Fisher Scientific, Gibco^TM, catalog number: 31985070 )
    
11. Nigericin sodium salt resuspended at 5 mg/ml in 100% ethanol (Sigma-Aldrich, catalog number: N7143 )
    
12. poly(dA:dT) resuspended at 1 mg/ml (InvivoGen, catalog number: tlrl-patn-1 )
    
13. Lipofectamine 2000 (Thermo Fischer Scientific, Invitrogen^TM, catalog number: 11668019 )
    
14. EDTA (Acros Organics, catalog number: 118432500 )
    
15. Disuccinimidyl suberate (DSS) (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 21655 )
    
16. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: 41640 )
    
17. Rabbit anti-ASC antibody (Santa Cruz Biotechnology, catalog number: sc-22514-R or Martin Oeggerli, Adipogen, catalog number: AG-25B-0006-C100)
    
18. Peroxidase-conjugated goat anti-Rabbit IgG (H+L) (Jackson ImmunoResearch, catalog number: 115-035-146 )
    
19. Nelfinavir Mesylate 10 mM in DMSO (Axon Medchem, catalog number: AG-1342 )
    
20. ECL Western blotting detection reagent (GE Healthcare, catalog number: RPN2106 )
    
21. Sodium chloride (NaCl)
    
22. Potassium chloride (KCl) (AppliChem, catalog number: A1362 )
    
23. Sodium phosphate dibasic (Na₂HPO₄) (AppliChem, catalog number: 131965.1210 )
    
24. Potassium dihydrogen phosphate (KH₂PO₄) (AppliChem, catalog number: A1042 )
    
25. RPMI 1640 (Thermo Fisher Scientific, Gibco^TM, catalog number: 681870010 )
    
26. Fetal bovine serum (FBS) (Thermo Fisher Scientific, Gibco^TM, catalog number: 10270106 )
    
27. Penicillin and streptomycin (Thermo Fisher Scientific, Gibco^TM, catalog number: 15640055 )
    
28. HEPES-KOH (BioConcept, Amimed, catalog number: 5-31F00-H )
    
29. Magnesium chloride (MgCl₂) (Sigma-Aldrich, catalog number: M8266 )
    
30. EGTA (Sigma-Aldrich, catalog number: 03777 )
    
31. Sucrose (Sigma-Aldrich, catalog number: 84097 )
    
32. PMSF (Sigma-Aldrich, catalog number: P7626 )
    
33. CHAPS (AppliChem, catalog number: A1099 )
    
34. SDS 20% (AppliChem, catalog number: A3942 )
    
35. Glycerol (AppliChem, catalog number: A0970 )
    
36. Bromophenol blue (Sigma-Aldrich, catalog number: B0126 )
    
37. Acrylamide (Applichem, catalog number: A1672 )
    
38. TEMED (AppliChem, catalog number: A1148 )
    
39. Ammonium persulfate (APS) (GE Healthcare, catalog number: 17-1311-01 )
    
40. Tris base (Biosolve, catalog number: 20092391 )
    
41. Ethanol (Fisher Scientific, catalog number: 10437341 )
    
42. Hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: 84422 )
    Note: This product has been discontinued.
    
43. Tween-20 (Applichem, catalog number: A1389 )
    
44. Non-fat dry milk 5% (Migros, Rapilait)
    
45. Lipopolysaccharide (LPS) resuspended at 5 mg/ml in endotoxin free water (InvivoGen, catalog number: tlrl-eklps )
    
46. 1x PBS (see Recipes)
    
47. Growth media (see Recipes)
    
48. Buffer A (see Recipes)
    
49. CHAPS buffer (see Recipes)
    
50. 4x protein loading buffer (see Recipes)
    
51. 15% SDS-PAGE (see Recipes)
    
52. Stacking gel (see Recipes)
    
53. Migration buffer SDS-PAGE (see Recipes)
    
54. Transfer buffer (see Recipes)
    
55. PBS-Tween (PBS-T) (see Recipes)
    
56. Blocking buffer (see Recipes)
    

Equipment

1. Pipettes (Gilson, Pipetman Classic)
   
2. Pipette aid (INTEGRA Biosciences, model: PIPETBOY acu 2 )
   
3. 37 °C, 5% CO₂ cell culture incubator (Thermo Fischer Scientific, Thermo Scientific^TM, model: Series 8000 Direct-Heat CO₂ Incubators )
   
4. Bench top refrigerated centrifuge for 1.5 ml tubes (LabNet International, model: Prism^TM R )
   
5. Tissue culture class II laminar flow hood (Gelaire, model: TC48 )
   
6. SDS-PAGE Migration System (VWR, Peqlab, model: PerfectBlue^TM Dual Gel System Twin ExWS )
   
7. Electrotransfer Blotter System (VWR, Peqlab, model: PerfectBlue^TM Tank Electro Blotter )
   

Procedure

1. Sample preparation and cross-linking
   1. At 18 h before stimulation, seed macrophages in 6-well plates containing 2 ml growth media in each well, 1 well per condition (in simplicate), at a density of 1.5 x 10⁶ cells per well.
      
   2. Wash cells twice with PBS and prime cells with 1 μg/ml LPS for 2 h in 1 ml Opti-MEM.
      
   3. Wash cells twice with PBS, and activate inflammasome by stimulating cells with 5 μM nigericin for 30 min or 25 μM nelfinavir for 6 h or transfect for 4 h with 2 μg/ml poly(dA:dT) using Lipofectamine 2000 (use DNA/Lipofectamine 2000 in the ratio of 1 μg/2 μl) in 1 ml Opti-MEM.
      
   4. Optional: harvest supernatants to assess the release of mature IL-1β and caspase-1 cleavage by Western blot.
      Note: Proteins from the supernatants can be precipitated using methanol/chloroform. If required supernatants can be stored frozen and proteins precipitated later. Precipitated proteins resuspended in 2x protein loading buffer can also be stored frozen for further Western blotting.
      
   5. Detach cells by scraping in 1 ml ice cold PBS containing 2 mM EDTA and centrifuge for 5 min at 1,500 x g at 4 °C.
      
   6. Resuspend cell pellets in 0.5 ml of ice-cold buffer A and lyse by shearing 30 times through a 21-gauge needle in microcentrifuge tubes. Centrifuge lysates in 1.5 ml Eppendorf tubes for 8 min at 1,800 x g, 4 °C to remove bulk nuclei. Keep 30 μl of lysates for Western blots of ASC as input controls.
      
   7. Dilute the remaining supernatants in a 1:1 ratio with buffer A and centrifuge at 2,000 x g for 5 min at 4 °C. The dilution is required to optimize harvest.
      
   8. After centrifugation, collect and dilute supernatants with 1 volume of CHAPS buffer and centrifuge at 5,000 x g for 8 min to pellet ASC oligomers.
      
   9. Discard supernatants and resuspend pellets in 50 μl of CHAPS buffer containing 4 mM of disuccinimidyl suberate (dissolved in DMSO). Incubate for 30 min at room temperature to cross-link proteins.
      
   10. Centrifuge at 5,000 x g for 8 min at 4 °C, discard supernatants and resuspend pellets in 30 μl of 2x protein loading buffer. Heat the samples for 2 min at 90 °C.
       Note: It is possible to stop at this point and freeze protein lysates at -20 °C before continuing the protocol with Western blotting steps.
       
       
2. Western blot analysis
   1. Assemble a 15% SDS-PAGE.
      Note: A 15% gel with a distance of migration of 7 to 8 cm is sufficient to give a good resolution of the ASC monomers and oligomers.
      
   2. Load 30 μl of resuspended ASC containing pellets on the 15% SDS-PAGE. (This sample should contain the cross-linked oligomerized ASC)
      
   3. Add 30 μl of protein loading buffer to 30 μl of input isolated during step A6, heat the samples for 2 min at 90 °C and load 30 μl on 15% SDS-PAGE. (This sample should contain the input ASC amounts)
      
   4. Separate at constant 160 V until protein loading buffer reaches the end of the gel.
      
   5. Transfer at 80 V for 2 h using a wet transfer system.
      
   6. Rinse membrane with distilled water.
      
   7. Block membrane in blocking buffer for 1 h at room temperature with agitation.
      
   8. Incubate membrane overnight at 4 °C with gentle agitation with anti-ASC antibody diluted 1:1,000 in blocking buffer.
      
   9. Wash membrane 3 times for 10 min with PBS-T (each wash).
      
   10. Incubate for 1 h with a 1:5,000 dilution of secondary antibody in blocking buffer at room temperature with agitation.
       
   11. Wash membrane 3 times for 10 min with PBS-T (each wash).
       
   12. Remove excess of PBS-T.
       
   13. Incubate with ECL and proceed to detection of chemiluminescence.
       

Data analysis

Immunoreactive bands for ASC oligomers appear at molecular weights corresponding to ASC monomers, dimers, and higher orders of oligomers. A positive control for ASC expression in the total cell lysates of each condition needs to be done. Other positive controls for equal protein loading such as tubulin can be included. Three independent experiments should be done. An example of data analysis can be found in Figure 1 and in Figure 3A of (Di Micco et al., 2016). In this study immortalized bone marrow-derived macrophages (iBMDMs) left unchallenged (lane 1) or challenged with NLRP3 inflammasome activator Nigericin (lane 2) or AIM2 inflammasome activators poly(dA:dT) (lane 3) and Nelfinavir (lane 4). In the first lane, resting iBMDMs did not exhibit ASC oligomerization in the pelleted fraction. In lanes 2, 3 and 4, iBMDMs challenged with inflammasome activators exhibited strong ASC oligomerization in their cross-linked pellets.


Figure 1. BMDMs were primed with LPS and treated for 6 h with vehicle, DMSO (Lane1) Nelfinavir, (Lane 2), Lipofectamine (Lipo) (lane 3) Lipofectamine plus poly(dA:dT) (Lane 4), as indicated. Cross-linked pellets (Pell) or soluble lysates (Lys) were immunoblotted for ASC or caspase-1. More details on the experiment can be found in Di Micco et al., 2016.

Notes

1. Proteins from cell culture supernatants should be precipitated and loaded on 15% SDS-PAGE in order to simultaneously determine the release of mature IL-1β and caspase-1 cleavage to ASC oligomerization.
   
2. All stimulations are performed in Opti-MEM without serum. Presence of serum in culture media will drastically interfere with Western blotting of proteins precipitated from cell culture supernatants.
   
3. LPS and Nigericin are toxic compounds that should be handled with care.
   
4. LPS is resuspended at 5 μg/μl in water and stored aliquoted at -20 °C for up to 12 months. Nigericin sodium salt is resuspended in EtOH at a concentration of 5 mM and conserved aliquoted at 4 °C for up to 12 months. Nelfinavir mesylate is resuspended in DMSO at 50 mM and stored in aliquots at -20 °C for up to 12 months. Poly(dA:dT) is resuspended in H₂O at 1 μg/μl and stored in aliquots at -20 °C for up to 12 months. All stimuli are made fresh from these stock solutions immediately before stimulations.
   

Recipes

1. 1x PBS
   137 mM NaCl
   2.7 mM KCl
   10 mM Na₂HPO₄
   1.8 mM KH₂PO₄
   
2. Growth media
   RPMI 1640
   10% FBS
   1% penicillin-streptomycin
   
3. Buffer A
   20 mM HEPES-KOH, pH 7.5
   10 mM KCl
   1.5 mM MgCl₂
   1 mM EDTA
   1 mM EGTA
   320 mM sucrose
   
4. CHAPS buffer
   20 mM HEPES-KOH, pH 7.5
   5 mM MgCl₂
   0.5 mM EGTA
   0.1 mM PMSF
   0.1% CHAPS
   
5. 4x protein loading buffer
   50 mM Tris-HCl, pH 6.8
   2% SDS
   10% glycerol
   12.5 mM EDTA
   0.02% bromophenol blue
   Note: 2x loading buffer is made by diluting the 4x protein loading buffer 2x in distilled water and does NOT contain reducing agent like DTT or 2-mercaptoethanol.
   
6. 15% SDS-PAGE
   375 mM Tris pH 8.8
   15% acrylamide
   1:1,000 (v:v) TEMED
   0.1% APS
   
7. Stacking gel
   125 mM Tris pH 6.8
   4.2% acrylamide
   1:1,000 (v:v) TEMED
   0.1% APS
   
8. Migration buffer SDS-PAGE
   25 mM Tris base, pH 8.3
   250 mM glycine
   0.1% SDS
   
9. Transfer buffer
   25 mM Tris base
   190 mM glycine
   20% ethanol
   Adjust pH to 8.3 with HCl
   
10. PBS-Tween (PBS-T)
    1x PBS
    0.1% Tween-20
    
11. Blocking buffer
    PBS-T
    Non-fat dry milk 5% (w/v)

Acknowledgments

This work was supported by European Research Council Starting Grant 281996 and is adapted from (Di Micco et al., 2016).

References

1. Dick, M. S., Sborgi, L., Ruhl, S., Hiller, S. and Broz, P. (2016). ASC filament formation serves as a signal amplification mechanism for inflammasomes. Nat Commun 7: 11929.
   
2. Di Micco, A., Frera, G., Lugrin, J., Jamilloux, Y., Hsu, E. T., Tardivel, A., De Gassart, A., Zaffalon, L., Bujisic, B., Siegert, S., Quadroni, M., Broz, P., Henry, T., Hrycyna, C. A. and Martinon, F. (2016). AIM2 inflammasome is activated by pharmacological disruption of nuclear envelope integrity. Proc Natl Acad Sci U S A 113(32): E4671-4680.
   
3. Fernandes-Alnemri, T., Wu, J., Yu, J. W., Datta, P., Miller, B., Jankowski, W., Rosenberg, S., Zhang, J. and Alnemri, E. S. (2007). The pyroptosome: a supramolecular assembly of ASC dimers mediating inflammatory cell death via caspase-1 activation. Cell Death Differ 14(9): 1590-1604.
   
4. Hoss, F., Rodriguez-Alcazar, J. F. and Latz, E. (2016). Assembly and regulation of ASC specks. Cell Mol Life Sci.
   
5. Martinon, F., Burns, K. and Tschopp, J. (2002). The inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of proIL-β. Mol Cell 10(2): 417-426.
   
6. Sharma, D. and Kanneganti, T. D. (2016). The cell biology of inflammasomes: Mechanisms of inflammasome activation and regulation. J Cell Biol 213(6): 617-629.