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32961
Inflammasome Antibody Sampler Kit

Inflammasome Antibody Sampler Kit #32961

Western Blotting Image 1

Western blot analysis of extracts from mouse bone marrow-derived dendritic cells (BMDC) and various cell lines using NLRP3 (D4D8T) Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).

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Western Blotting Image 2

Western blot analysis of extracts from Daudi, KARPAS-299, and L-540 cell lines using AIM2 (D5X7K) Rabbit mAb.

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Western Blotting Image 3

Western blot analysis of extracts from THP-1 and MUTZ-3 cells using NLRC4 (D5Y8E) Rabbit mAb.

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Western Blotting Image 4

Western blot analysis of extracts from THP-1 and NK-92 cells using Caspase-1 (D7F10) Rabbit mAb.

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Western Blotting Image 5

Western blot analysis of extracts from K-562 and SH-SY5Y cells and from rat testis using NALP1 Antibody.

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Western Blotting Image 6

Western blot analysis of extracts from various cell lines using TMS1 (E1E3I) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower). As expected, Jurkat cells do not express TMS1.

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Western Blotting Image 7

Western blot analysis of extracts from COS-7 cells, untransfected () or transfected with construct overexpressing human caspase-1 (+), using Cleaved Caspase-1 (Asp297) (D57A2) Rabbit mAb (upper) or Caspase-1 (D7F10) Rabbit mAb #3866 (lower).

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Western Blotting Image 8

Western blot analysis of recombinant Human Interleukin-1β (hIL-1β) #8900 using Cleaved-IL-1β (Asp116) (D3A3Z) Rabbit mAb.

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Western Blotting Image 9

Western blot analysis of extracts from THP-1 cells, untreated (-) or LPS-treated (100 ng/ml, 3 hr; +), using IL-1β (D3U3E) Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).

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Western Blotting Image 10

After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.

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IP Image 11

Immunoprecipitation of NLRP3 from J774A.1 cell extracts using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or NLRP3 (D4D8T) Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using NLRP3 (D4D8T) Rabbit mAb.

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Western Blotting Image 12

Western blot analysis of extracts from HL-60 cells, serum-starved and either untreated (-) or treated overnight with Human Interferon-γ (hIFN-γ) #8901 (100 ng/ml; +), using AIM2 (D5X7K) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).

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Western Blotting Image 13

Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing full-length human NLRC4 (hNLRC4; +), using NLRC4 (D5Y8E) Rabbit mAb.

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Western Blotting Image 14

Western blot analysis of extracts from COS cells, untransfected or transfected with human caspase-1, using Caspase-1 (D7F10) Rabbit mAb.

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IP Image 15

Immunoprecipitation of TMS1 from THP-1 cell extracts using Rabbit (D1AE) mAb XP® Isotype Control #3900 (lane 2) or TMS1 (E1E3I) Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot was performed using TMS1 (E1E3I) Rabbit mAb.

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Western Blotting Image 16

Western blot analysis of extracts from the media of THP-1 cells, differentiated with TPA #9905 (80 nM, overnight) followed by treatment with LPS (1 mu-g/ml, 8 hours), using Cleaved Caspase-1 (Asp297) (D57A2) Rabbit mAb.

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Western Blotting Image 17

Western blot analysis of extracts from cells or media collected from THP-1 cells, differentiated with TPA #4147 (80 nM, overnight) and subsequently treated with (+) or without (-) Lipopolysaccharides (LPS) #14011 (1 μg/ml, 6 hr), using Cleaved-IL-1β (Asp116) (D3A3Z) Rabbit mAb.

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Western Blotting Image 18

Western blot analysis of recombinant Human Interleukin-1β (hIL-1β) #8900 using IL-1β (D3U3E) Rabbit mAb.

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Western Blotting Image 19

Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing Myc-DDK tagged human AIM2 (hAIM2-Myc/DDK; +), using AIM2 (D5X7K) Rabbit mAb (upper) and Myc-Tag (71D10) Rabbit mAb #2278 (lower).

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IP Image 20

Immunoprecipitation of NLRC4 from MUTZ-3 cell extracts using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or NLRC4 (D5Y8E) Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using NLRC4 (D5Y8E) Rabbit mAb.

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IP Image 21

Immunoprecipitation of Cleaved-IL-1β (Asp116) from extracts of THP-1 cells differentiated with TPA #4147 (80 nM, overnight) followed by treatment with Lipopolysaccharides (LPS) #14011 (1 μg/ml, 6 hr). Lane 1 is 10% input, lane 2 is precipitated with Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is precipitated with Cleaved-IL-1β (Asp116) (D3A3Z) Rabbit mAb. Western blot was performed using Cleaved-IL-1β (Asp116) (D3A3Z) Rabbit mAb.

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Flow Cytometry Image 22

Flow cytometric analysis of THP-1 cells, untreated (blue) or LPS-treated (100 ng/ml, 3 hr; green), using IL-1β (D3U3E) Rabbit mAb. Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 647 Conjugate) #4414 was used as a secondary antibody.

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IP Image 23

Immunoprecipitation of AIM2 from HL-60 cell extracts treated with Human Interferon-γ (hIFN-γ) #8901 (100 ng/ml, overnight) using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or AIM2 (D5X7K) Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot was performed using AIM2 (D5X7K) Rabbit mAb.

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IF-IC Image 24

Confocal immunofluorescent analysis of THP-1 cells, differentiated with TPA #4174 (80 nM, 24 hr) and subsequently treated with (right) or without (left) Lipopolysaccharides (LPS) #14011 (1 μg/ml, 6 hr), using Cleaved-IL-1β (Asp116) (D3A3Z) Rabbit mAb (green). Actin filaments were labeled with DyLight™ 554 Phalloidin #13054 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

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IF-IC Image 25

Confocal immunofluorescent analysis of THP-1 cells, untreated (left) or LPS-treated (500 ng/ml, 2 hr; right), using IL-1β (D3U3E) Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red).

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Product Includes Quantity Applications Reactivity MW(kDa) Isotype
NLRP3 (D4D8T) Rabbit mAb 15101 20 µl
  • WB
  • IP
H M 110 Rabbit IgG
AIM2 (D5X7K) Rabbit mAb 12948 20 µl
  • WB
  • IP
H 40 Rabbit IgG
NLRC4 (D5Y8E) Rabbit mAb 12421 20 µl
  • WB
  • IP
H 110 Rabbit IgG
Caspase-1 (D7F10) Rabbit mAb 3866 20 µl
  • WB
  • IP
H 48, 20 Rabbit IgG
NALP1 Antibody 4990 20 µl
  • WB
H M R 165, 70 Rabbit 
TMS1 (E1E3I) Rabbit mAb 13833 20 µl
  • WB
  • IP
H 22, 19, 15 Rabbit IgG
Cleaved Caspase-1 (Asp297) (D57A2) Rabbit mAb 4199 20 µl
  • WB
  • IP
H 20, 22 Rabbit IgG
Cleaved-IL-1β (Asp116) (D3A3Z) Rabbit mAb 83186 20 µl
  • WB
  • IP
  • IF
H 17 Rabbit IgG
IL-1β (D3U3E) Rabbit mAb 12703 20 µl
  • WB
  • IF
  • F
H 17, 31 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 

The Inflammasome Antibody Sampler Kit provides an economical means of detecting multiple inflammasome components. The kit contains enough primary antibodies to perform at least two western blot experiments.

Each antibody in the Inflammasome Antibody Sampler Kit detects endogenous levels of its target protein. AIM2 (D5X7K) Rabbit mAb detects a 22 kDa band of unknown origin in some cell lines. Caspase-1 (D7F10) Rabbit mAb detects endogenous levels of full-length human Caspase-1; the activated p20 subunit was detected by over-expression. NALP1 Antibody detects endogenous levels of total NALP1 protein and also detects a 70 kDa protein that correlates with a predicted short form (NALP1s) that lacks the leucine repeat region. TMS1 (E1E3I) Rabbit mAb can detect three known isoforms of TMS1. Cleaved Caspase-1 (Asp297) (D57A2) Rabbit mAb detects endogenous levels of the p20 subunit of human caspase-1 only upon cleavage at Asp297. Cleaved-IL-1β (Asp116) (D3A3Z) Rabbit mAb recognizes endogenous levels of mature IL-1β protein only when cleaved at Asp116. IL-1β (D3U3E) Rabbit mAb is not able to detect endogenous levels of mature IL-1β. It can detect up to 100 pg of recombinant mature IL-1β.

Monoclonal and polyclonal antibodies are produced by immunizing animals with recombinant human IL-1β protein or with synthetic peptides corresponding to residues adjacent to Asp297 human caspase-1, residues adjacent to Asp116 of human IL-1β, residues surrounding Ala306 of mouse NLRP3, Lys93 of human AIM2, Leu942 of human NLRC4, Gly1081 of human NALP1, and the carboxy terminus of human TMS1 isoform 1. Polyclonal antibodies are purified by protein A and peptide affinity chromatography.

The innate immune system works as the first line of defense in protection from pathogenic microbes and host-derived signals of cellular distress. One way in which these “danger” signals trigger inflammation is through activation of inflammasomes, which are multiprotein complexes that assemble in the cytosol after exposure to pathogen-associated molecular patterns (PAMPs) or danger-associated molecular patterns (DAMPs) and result in the activation of caspase-1 and subsequent cleavage of proinflammatory cytokines IL-1β and IL-18 (Reviewd in 1-6). Inflammasome complexes typically consist of a cytosolic pattern recognition receptor (PRR; a nucleotide-binding domain and leucine-rich-repeat [NLR] or AIM2-like receptor [ALR] family member), an adaptor protein (ASC/TMS1), and pro-caspase-1. A number of distinct inflammasome complexes have been identified, each with a unique PRR and activation triggers. The best characterized is the NLRP3 complex, which contains NLRP3, ASC, and pro-caspase-1. The NLRP3 inflammasome is activated in a two-step process. First, NF-κB signaling is induced through PAMP- or DAMP-mediated activation of TLR4 or TNFR, resulting in increased expression of NLRP3, pro-IL-1β, and pro-IL-18 (priming step, signal 1). Next, indirect activation of NLRP3 occurs by a multitude of signals (whole pathogens, PAMPs/DAMPs, potassium efflux, lysosomal-damaging environmental factors [uric acid, silica, alum] and endogenous factors [amyloid-β, cholesterol crystals], and mitochondrial damage), leading to complex assembly and activation of caspase-1 (signal 2). The complex inflammasome structure is built via domain interactions among the protein components. Other inflammasomes are activated by more direct means: double-stranded DNA activates the AIM2 complex, anthrax toxin activates NLRP1, and bacterial flagellin activates NLRC4. Activated caspase-1 induces secretion of proinflammatory cytokines IL-1β and -18, but also regulates metabolic enzyme expression, phagosome maturation, vasodilation, and pyroptosis, an inflammatory programmed cell death. Inflammasome signaling contributes to the onset of a number of diseases, including atherosclerosis, type II diabetes, Alzheimer’s disease, and autoimmune disorders.

  1. Broz, P. and Dixit, V.M. (2016) Nat Rev Immunol 16, 407-20.
  2. Guo, H. et al. (2015) Nat Med 21, 677-87.
  3. Jo, E.K. et al. (2016) Cell Mol Immunol 13, 148-59.
  4. Rathinam, V.A. and Fitzgerald, K.A. (2016) Cell 165, 792-800.
  5. Shao, B.Z. et al. (2015) Front Pharmacol 6, 262.
  6. Schroder, K. and Tschopp, J. (2010) Cell 140, 821-32.
Entrez-Gene Id
9447 , 29108 , 834 , 3553 , 22861 , 58484 , 216799
Swiss-Prot Acc.
O14862 , Q9ULZ3 , P29466 , P01584 , Q9C000 , Q9NPP4 , Q8R4B8
For Research Use Only. Not For Use In Diagnostic Procedures.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.

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