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Detection of ASC Oligomerization by Western Blotting
蛋白印迹法检测ASC寡聚化   

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Abstract

The apoptosis-associated speck-like protein with a caspase-recruitment domain (ASC) adaptor protein bridges inflammasome sensors and caspase-1. Upon inflammasome activation, ASC nucleates in a prion-like manner into a large and single platform responsible for the recruitment and the activation of caspase-1. Active caspase-1 will in turn promote the proteolytic maturation of the pro-inflammatory cytokine IL-1β. ASC oligomerization is a direct evidence for inflammasome activation and its detection allows a read-out independent of caspase-1 and IL-1β. This protocol describes how to detect the oligomerization of ASC by Western blot.

Keywords: Inflammasome(炎性小体), Caspase-1(半胱天冬酶-1), Pyroptosis(细胞焦亡), Biochemistry(生物化学), Auto-inflammation(自身炎症)

Background

Inflammasomes are large multiprotein platforms that sense a variety of microbial, endogenous and environmental stressors leading to the maturation of the pro-inflammatory IL-1 family of cytokines (Martinon et al., 2002; Sharma and Kanneganti, 2016). Upon activation, inflammasome sensors recruit the adaptor protein ASC through pyrin domain (PYD)-PYD homotypic interactions. ASC will in turn bind to caspase-1 via caspase activation and recruitment domain (CARD)-CARD interactions and favor auto-proteolytic cleavage of caspase-1, leading to maturation of IL-1β and IL-18 (Hoss et al., 2016). Inflammasome activation triggers supramolecular oligomerization of ASC dimers into large interweaving fibrils also termed ‘ASC-specks’ or ‘pyroptosome’ (Fernandes-Alnemri et al., 2007). ASC-speck/pyroptosomes is a hallmark of inflammasome activation that correlates with caspase-1 cleavage and release of mature IL-1β (Dick et al., 2016). Recently we showed that Nelfinavir, an HIV-protease inhibitor, promotes the release of self-DNA into the cytosol, activates the DNA sensing inflammasome AIM2 and subsequent ASC oligomerization (Di Micco et al., 2016). This protocol aims at detecting endogenous ASC oligomerization in immortalized bone marrow-derived macrophages (iBMDMs) by Western blot analysis. It is adapted from the publication of Fernandes-Alnemri et al. (2007) that used this technique to detect ASC oligomerization in THP-1 cells.

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, GibcoTM, 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, InvitrogenTM, catalog number: 11668019 )
  14. EDTA (Acros Organics, catalog number: 118432500 )
  15. Disuccinimidyl suberate (DSS) (Thermo Fisher Scientific, Thermo ScientificTM, 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, GibcoTM, catalog number: 681870010 )
  26. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10270106 )
  27. Penicillin and streptomycin (Thermo Fisher Scientific, GibcoTM, 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 ScientificTM, model: Series 8000 Direct-Heat CO2 Incubators )
  4. Bench top refrigerated centrifuge for 1.5 ml tubes (LabNet International, model: PrismTM R )
  5. Tissue culture class II laminar flow hood (Gelaire, model: TC48 )
  6. SDS-PAGE Migration System (VWR, Peqlab, model: PerfectBlueTM Dual Gel System Twin ExWS )
  7. Electrotransfer Blotter System (VWR, Peqlab, model: PerfectBlueTM 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.

简介

具有半胱天冬酶募集区(ASC)衔接蛋白的凋亡相关斑点样蛋白桥联炎症体传感器和半胱天冬酶-1。在炎性体活化后,ASC以类似朊病毒的方式成核,成为负责募集和激活半胱天冬酶-1的大而单一的平台。活性胱天蛋白酶-1将反过来促进促炎细胞因子IL-1β的蛋白水解成熟。 ASC寡聚化是炎性体激活的直接证据,其检测允许读取与caspase-1和IL-1β无关。该方案描述了如何通过蛋白质印迹检测ASC的寡聚化。

背景 Inflammasomes是大量的多蛋白平台,其感测各种微生物,内源和环境胁迫因子,导致促炎IL-1细胞因子家族的成熟(Martinon等人,2002; Sharma和Kanneganti, 2016)。激活后,炎性细胞传感器通过pyrin结构域(PYD)-PYD同型相互作用募集衔接蛋白ASC。 ASC通过胱天蛋白酶激活和募集域(CARD)-CARD相互作用又结合半胱天冬酶-1,并有利于caspase-1的自我蛋白水解切割,导致IL-1β和IL-18的成熟(Hoss等人。,2016)。 Inflammasome激活引发ASC二聚体的超分子寡聚化成称为“ASC-specks”或“pyroptosome”(Fernandes-Alnemri等人,2007)的大交织原纤维。 ASC-speck / pyroptosomes是与caspase-1切割和成熟IL-1β的释放相关的炎性体激活的标志(Dick等人,2016)。最近我们显示奈非那韦是一种HIV-蛋白酶抑制剂,促进自身DNA释放到细胞溶质中,激活DNA感染炎性细胞AIM2和随后的ASC寡聚化(Di Micco等人,2016)。该方案旨在通过蛋白质印迹分析检测永生化骨髓来源的巨噬细胞(iBMDM)中的内源性ASC寡聚化。改编自Fernandes-Alnemri等人(2007)的出版物,其使用这种技术来检测THP-1细胞中的ASC寡聚化。

关键字:炎性小体, 半胱天冬酶-1, 细胞焦亡, 生物化学, 自身炎症

材料和试剂

  1. 6个培养皿(TPP,目录号:92406)
  2. 注射器1 ml(BD,目录号:300013)
  3. 21号针(B.Braun Melsungen,Sterican ®,目录号:4657527)
  4. 1.5ml Eppendorf管(Corning,Axygen ,目录号:MCT-150-C-S)
  5. 200μl移液器吸头(STARLAB INTERNATIONAL,TipOne,目录号:S1111-1000)
  6. 1,000μl移液器吸头(STARLAB INTERNATIONAL,TipOne,目录号:S1111-6001)
  7. 细胞刮刀(Corning,Falcon ®,目录号:352340)
  8. 硝酸纤维素印迹膜(Amersham)(GE Healthcare,目录号:10600003)
  9. 来自瑞士巴塞尔大学Biozentrum的Petr Broz教授获得的永生性小鼠骨髓衍生巨噬细胞(iBMDM)
  10. Opti-MEM(Thermo Fisher Scientific,Gibco TM,目录号:31985070)
  11. 尼日利亚钠盐以5mg/ml在100%乙醇中重悬(Sigma-Aldrich,目录号:N7143)
  12. 聚(dA:dT)以1mg/ml重悬(InvivoGen,目录号:tlrl-patn-1)
  13. Lipofectamine 2000(Thermo Fischer Scientific,Invitrogen TM,目录号:11668019)
  14. EDTA(Acros Organics,目录号:118432500)
  15. 辛磺酸二琥珀酰亚胺酯(DSS)(Thermo Fisher Scientific,Thermo Scientific TM,目录号:21655)
  16. 二甲基亚砜(DMSO)(Sigma-Aldrich,目录号:41640)
  17. 兔抗ASC抗体(Santa Cruz Biotechnology,目录号:sc-22514-R或Martin Oeggerli,Adipogen,目录号:AG-25B-0006-C100)
  18. 过氧化物酶缀合的山羊抗兔IgG(H + L)(Jackson ImmunoResearch,目录号:115-035-146)
  19. Nelfinavir Mesylate 10 mM in DMSO(Axon Medchem,目录号:AG-1342)
  20. ECL Western印迹检测试剂(GE Healthcare,目录号:RPN2106)
  21. 氯化钠(NaCl)
  22. 氯化钾(KCl)(AppliChem,目录号:A1362)
  23. 磷酸氢二钠(Na 2 HPO 4)(AppliChem,目录号:131965.1210)
  24. 磷酸二氢钾(KH 2 PO 4)(AppliChem,目录号:A1042)
  25. RPMI 1640(Thermo Fisher Scientific,Gibco TM,目录号:681870010)
  26. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM,目录号:10270106)
  27. 青霉素和链霉素(Thermo Fisher Scientific,Gibco TM,目录号:15640055)
  28. HEPES-KOH(BioConcept,Amimed,目录号:5-31F00-H)
  29. 氯化镁(MgCl 2)(Sigma-Aldrich,目录号:M8266)
  30. EGTA(Sigma-Aldrich,目录号:03777)
  31. 蔗糖(Sigma-Aldrich,目录号:84097)
  32. PMSF(Sigma-Aldrich,目录号:P7626)
  33. CHAPS(AppliChem,目录号:A1099)
  34. SDS 20%(AppliChem,目录号:A3942)
  35. 甘油(AppliChem,目录号:A0970)
  36. 溴苯酚蓝(Sigma-Aldrich,目录号:B0126)
  37. 丙烯酰胺(Applichem,目录号:A1672)
  38. TEMED(AppliChem,目录号:A1148)
  39. 过硫酸铵(APS)(GE Healthcare,目录号:17-1311-01)
  40. Tris碱(Biosolve,目录号:20092391)
  41. 乙醇(Fisher Scientific,目录号:10437341)
  42. 盐酸(HCl)(Sigma-Aldrich,目录号:84422)
    注意:本产品已停产。
  43. 吐温-20(申请,目录号:A1389)
  44. 不含脂肪的干乳5%(Migros,Rapilait)
  45. 脂多糖(LPS)以5mg/ml重新悬浮于无内毒素游离水中(InvivoGen,目录号:tlrl-eklps)
  46. 1x PBS(见食谱)
  47. 成长媒介(见食谱)
  48. 缓冲液A(参见食谱)
  49. CHAPS缓冲区(见配方)
  50. 4x蛋白加载缓冲液(参见食谱)
  51. 15%SDS-PAGE(参见食谱)
  52. 堆叠凝胶(参见食谱)
  53. 迁移缓冲液SDS-PAGE(见配方)
  54. 转移缓冲区(见配方)
  55. PBS-Tween(PBS-T)(参见食谱)
  56. 阻塞缓冲区(见配方)

设备

  1. 移液器(Gilson,Pipetman Classic)
  2. 移液器辅助(INTEGRA Biosciences,型号:PIPETBOY acu 2)
  3. 37℃,5%CO 2细胞培养箱(Thermo Fischer Scientific,Thermo Scientific TM,型号:8000系列直接热CO 2系列孵化器)
  4. 用于1.5ml管的台式冷冻离心机(LabNet International,型号:Prism TM)
  5. 组织培养二级层流罩(Gelaire,型号:TC48)
  6. SDS-PAGE迁移系统(VWR,Peqlab,型号:PerfectBlue TM双重凝胶系统双ExWS)
  7. 电转印机系统(VWR,Peqlab,型号:PerfectBlue TM Tank Electro Blotter)

程序

  1. 样品制备和交联
    1. 在刺激前18小时,种子巨噬细胞在每个孔中含有2ml生长培养基的6孔板中,每孔条件(简单)为1孔,密度为每孔1.5×10 6个细胞。
    2. 用PBS洗涤细胞两次,并在1ml Opti-MEM中用1μg/ml LPS补充细胞2小时
    3. 用PBS洗涤细胞两次,通过用5μM尼日利昔酮刺激细胞30分钟或25μM奈非那韦6小时或用2μg/ml聚(dA:dT)转染4小时,使用Lipofectamine 2000(使用DNA/Lipofectamine 2000,比例为1μg/2μl)在1 ml Opti-MEM中
    4. 任选的:收获上清以评估成熟IL-1β和半胱天冬酶-1裂解通过蛋白质印迹的释放 注意:来自上清液的蛋白质可以使用甲醇/氯仿沉淀。如果需要上清液可以冷冻储存,蛋白质沉淀。重悬于2x蛋白加载缓冲液中的沉淀蛋白也可以冷冻保存以进行进一步的Western印迹。
    5. 通过在含有2mM EDTA的1ml冰冷的PBS中刮擦来分离细胞,并在4℃以1,500×g离心5分钟。
    6. 将细胞沉淀重悬于0.5ml冰冷缓冲液A中,并通过微量离心管中的21号针头剪切30次。在1.5ml Eppendorf管中离心裂解物,在1,800×g,4℃下静置8分钟以除去体积核。保留30μl的裂解液用于Western印迹的ASC作为输入对照
    7. 用缓冲液A以1:1的比例稀释剩余的上清液,并在4℃下以2,000xg离心5分钟。需要稀释来优化收获。
    8. 离心后,收集并稀释上清液与1体积的CHAPS缓冲液,并以5,000×g离心8分钟,以沉淀ASC寡聚体。
    9. 丢弃上清液并将沉淀重悬于50μl含有4mM二琥珀酰亚氨基辛酸酯(溶于DMSO中)的CHAPS缓冲液中。在室温下孵育30分钟以交联蛋白质。
    10. 在4℃下以5,000×g离心8分钟,弃去上清液,并将沉淀重悬于30μl的2x蛋白质加载缓冲液中。在90℃下将样品加热2分钟 注意:在此之前可以停止,并将蛋白裂解物冷冻至-20°C,然后继续使用Western印迹步骤。

  2. 蛋白质印迹分析
    1. 组装15%SDS-PAGE。
      注意:具有7至8厘米迁移距离的15%凝胶足以给出ASC单体和低聚物的良好拆分。
    2. 在15%SDS-PAGE上载入30μl重悬浮的含有ASC的沉淀物。 (此样本应包含交联的低聚ASC)
    3. 向步骤A6分离的30μl输入中加入30μl蛋白质加载缓冲液,在90℃下加热样品2分钟,并在15%SDS-PAGE上加载30μl。 (此样本应包含输入ASC量)
    4. 在恒定的160 V下分离,直到蛋白质加载缓冲液达到凝胶的末端。
    5. 使用湿式转移系统在80 V下转移2 h。
    6. 用蒸馏水冲洗膜。
    7. 阻塞缓冲液中的膜在室温下搅拌1小时
    8. 在4℃下孵育膜过夜,轻轻搅拌,用阻断缓冲液1:1,000稀释的抗ASC抗体
    9. 用PBS-T(每次洗涤)洗涤膜3次10分钟。
    10. 在室温下搅拌,在封闭缓冲液中孵育1小时,再用1:5,000稀释的二抗
    11. 用PBS-T(每次洗涤)洗涤膜3次10分钟。
    12. 去除多余的PBS-T。
    13. 与ECL孵育并进行化学发光检测。

数据分析

ASC寡聚体的免疫反应带以对应于ASC单体,二聚体和更高级寡聚体的分子量出现。需要对每个条件的总细胞裂解物中ASC表达的阳性对照进行。可以包括其他用于相等蛋白质负载的阳性对照如微管蛋白。应进行三次独立实验。数据分析的一个例子可以在图1和(Di Micco等人,2016)的图3A中找到。在本研究中,永生化的骨髓来源的巨噬细胞(iBMDM)未经挑战(泳道1)或用NLRP3炎性体激活物尼尼西肽(泳道2)或AIM2炎性体激活剂聚(dA:dT)(泳道3)和奈非那韦(泳道4)进行攻击。在第一条泳道中,休息的iBMDM在颗粒状部分中没有显示ASC寡聚化。在泳道2,3和4中,用炎性体激活剂攻击的iBMDM在其交联的小球中表现出强烈的ASC寡聚化。


图1. BMDM用LPS引发,用载体,DMSO(Lane1)奈非那韦,(泳道2),脂质体(Lipo)(泳道3)Lipofectamine加聚(dA:dT)(泳道4)处理6小时, ,如所示。将交联小球(Pell)或可溶性裂解物(Lys)免疫印迹为ASC或半胱天冬酶-1。关于实验的更多细节可以在Di Micco等人,2016中找到。

笔记

  1. 应将细胞培养上清液中的蛋白质沉淀并加载到15%SDS-PAGE上,以同时测定成熟IL-1β和半胱天冬酶-1切割至ASC寡聚化的释放。
  2. 所有刺激均在无血清的Opti-MEM中进行。在培养基中血清的存在会大大干扰从细胞培养上清液中沉淀的蛋白质的蛋白质印迹
  3. LPS和尼可地霉素是有毒化合物,应小心处理。
  4. 将LPS以5μg/μl重悬于水中,并在-20℃下等分至少12个月。将尼日利亚钠盐以5mM的浓度重悬于EtOH中,并在4℃保存长达12个月。将甲磺酸奈非那韦以50mM重悬于DMSO中,并以等分试样在-20℃保存长达12个月。将聚(dA:dT)以1μg/μl重悬于H 2 O中,并以等分试样在-20℃下储存长达12个月。所有刺激物在刺激前立即从这些储备溶液中清新。

食谱

  1. 1x PBS
    137 mM NaCl
    2.7 mM KCl
    10mM Na 2 HPO 4
    1.8mM KH PO 4
  2. 成长媒体
    RPMI 1640
    10%FBS
    1%青霉素 - 链霉素
  3. 缓冲区A
    20mM HEPES-KOH,pH7.5
    10 mM KCl
    1.5mM MgCl 2
    1 mM EDTA
    1 mM EGTA
    320毫克蔗糖
  4. CHAPS缓冲区
    20mM HEPES-KOH,pH7.5
    5mM MgCl 2
    0.5 mM EGTA
    0.1 mM PMSF
    0.1%CHAPS
  5. 4x蛋白加载缓冲液
    50mM Tris-HCl,pH6.8
    2%SDS
    10%甘油
    12.5 mM EDTA
    0.02%溴酚蓝
    注意:2x加载缓冲液是通过在蒸馏水中稀释4倍蛋白质加载缓冲液2x制成的,不含减少剂,如DTT或2-巯基乙醇。
  6. 15%SDS-PAGE
    375 mM Tris pH 8.8
    15%丙烯酰胺
    1:1,000(v:v)TEMED
    0.1%APS
  7. 堆叠凝胶
    125 mM Tris pH 6.8
    4.2%丙烯酰胺
    1:1,000(v:v)TEMED
    0.1%APS
  8. 迁移缓冲液SDS-PAGE
    25mM Tris碱,pH 8.3
    250mM甘氨酸
    0.1%SDS
  9. 转移缓冲区
    25 mM Tris碱基
    190mM甘氨酸
    20%乙醇
    用HCl调节pH至8.3
  10. PBS-Tween(PBS-T)
    1x PBS
    0.1%Tween-20
  11. 阻塞缓冲区
    PBS-T
    不含脂肪的干乳5%(w/v)

致谢

这项工作得到欧洲研究委员会起草授权281996的支持,并由(Di Micco等人,2016年)改编而成。

参考

  1. Dick,MS,Sborgi,L.,Ruhl,S.,Hiller,S.和Broz,P。(2016)。 ASC细丝形成用作炎症信号的信号放大机制。 7:11929.
  2. Di Micco,A.,Frera,G.,Lugrin,J.,Jamilloux,Y.,Hsu,ET,Tardivel,A.,De Gassart,A.,Zaffalon,L.,Bujisic,B.,Siegert, ,Quadroni,M.,Broz,P.,Henry,T.,Hrycyna,CA和Martinon,F。(2016)。< a class ="ke-insertfile"href ="http://www.ncbi。 nlm.nih.gov/pubmed/27462105"target ="_ blank"> AIM2炎症细胞被核包膜完整性的药理学破坏所激活。 Proc Natl Acad Sci USA 113(32):E4671 -4680。
  3. Fernandes-Alnemri,T.,Wu,J.,Yu,JW,Datta,P.,Miller,B.,Jankowski,W.,Rosenberg,S.,Zhang,J.and Alnemri,ES(2007) 焦虫体:通过介导炎症细胞死亡介导ASC二聚体的超分子组装半胱天冬酶-1激活。细胞死亡差异 14(9):1590-1604。
  4. Hoss,F.,Rodriguez-Alcazar,JF和Latz,E.(2016)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/27761594 "target ="_ blank"> ASC斑点的组装和调节。细胞分子生命科学。
  5. Martinon,F.,Burns,K.和Tschopp,J。(2002)。炎症体:触发炎症半胱天冬酶激活和proIL-β的加工的分子平台。分子细胞<10>(2):417-426。
  6. Sharma,D.和Kanneganti,TD(2016)。炎症细胞的细胞生物学:炎性体激活和调节的机制。细胞生物学 213(6):617-629。
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引用:Lugrin, J. and Martinon, F. (2017). Detection of ASC Oligomerization by Western Blotting. Bio-protocol 7(10): e2292. DOI: 10.21769/BioProtoc.2292.
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