蛋白印迹法检测ASC寡聚化

Jérôme  Lugrin; Fabio  Martinon

doi:10.21769/BioProtoc.2292

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Peer-reviewed

Detection of ASC Oligomerization by Western Blotting

蛋白印迹法检测ASC寡聚化

Jérôme Lugrin

F Fabio Martinon email

发布: 2017年05月20日第7卷第10期 DOI: 10.21769/BioProtoc.2292 浏览次数: 21972

评审: Andrea PuharSaskia F. ErttmannMeenal Sinha

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The authors used this protocol in:

Cover of Proceedings of the National Academy of Sciences of the United States of America, featuring study using the protocol.

Aug 2016

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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 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

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

Equipment

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