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20836
Mouse Reactive Inflammasome Antibody Sampler Kit
Primary Antibodies
Antibody Sampler Kit

Mouse Reactive Inflammasome Antibody Sampler Kit #20836

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Mouse Reactive Inflammasome Antibody Sampler Kit: Image 1

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.

Mouse Reactive Inflammasome Antibody Sampler Kit: Image 2

Immunoprecipitation of mouse AIM2 from J774A.1 cell extracts. Lane 1 is 10% input, lane 2 is Normal Rabbit IgG #2729, and lane 3 is AIM2 Antibody (Mouse Specific). Western blot was performed using AIM2 Antibody (Mouse Specific). Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb (HRP Conjugate) #5127 was used as a secondary antibody.

Mouse Reactive Inflammasome Antibody Sampler Kit: Image 3

Flow cytometric analysis of Raw264.7 cells (blue) and J774A.1 cells (green) using ASC/TMS1 (D2W8U) Rabbit mAb (Mouse Specific) (solid lines) or a concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.

Mouse Reactive Inflammasome Antibody Sampler Kit: Image 4

Immunoprecipitation of Cleaved-IL-1β (Asp117) from extracts of media from mouse bone marrow derived macrophages (mBMDM) treated with LPS #14011 (50 ng/ml, 4 hr) followed by nigericin (15 μM, 45 min). Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is Cleaved-IL-1β (Asp117) (E7V2A) Rabbit mAb (Mouse Specific). Western blot was performed using Cleaved-IL-1β (Asp117) (E7V2A) Rabbit mAb (Mouse Specific). Anti-Rabbit IgG, HRP-linked Antibody #7074 was used as a secondary antibody.

Mouse Reactive Inflammasome Antibody Sampler Kit: Image 5

Flow cytometric analysis of Raw264.7 cells, untreated (blue) or treated with LPS #14011 (100ng/ml, 6 hrs; green) using IL-1β (D6D6T) Rabbit mAb (Mouse Specific) (solid lines) or concentration matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.

Mouse Reactive Inflammasome Antibody Sampler Kit: Image 6

Immunoprecipitation of Cleaved Caspase-1 (Asp296) from extracts of acetone precipitated media from mouse bone marrow derived macrophages treated with Lipopolysaccharides (LPS) #14011 (50ng/ml, 4hr) followed by Nigericin (15 μM, 45 min). Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is Cleaved Caspase-1 (Asp296) (E2G2I) Rabbit mAb. Western blot analysis was performed using Cleaved Caspase-1 (Asp296) (E2G2I) Rabbit mAb. Anti-rabbit IgG, HRP-linked Antibody #7074 was used as a secondary antibody.

Mouse Reactive Inflammasome Antibody Sampler Kit: Image 7

Immunoprecipitation of Caspase-1 from EL4 cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 Caspase-1 (E2Z1C) Rabbit mAb. Western blot was performed using Caspase-1 (E2Z1C) Rabbit mAb. Mouse Anti-rabbbit IgG (Conformation Specific) (L27A9) mAb (HRP Conjugate) #5127 was used as a secondary antibody.

Mouse Reactive Inflammasome Antibody Sampler Kit: Image 8

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.

Mouse Reactive Inflammasome Antibody Sampler Kit: Image 9

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).

Mouse Reactive Inflammasome Antibody Sampler Kit: Image 10

Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing full-length mouse AIM2 (mAIM2; +), using AIM2 Antibody (Mouse Specific).

Mouse Reactive Inflammasome Antibody Sampler Kit: Image 11

Confocal immunofluorescent analysis of mouse Tg2576 brain which overexpresses mutant human APP695. Sections were first labeled with ASC/TMS1 (D2W8U) Rabbit mAb (Mouse Specific) #67824 (green) and APP/β-Amyloid (NAB228) Mouse mAb #2450 (yellow). After blocking free secondary binding sites with Mouse (G3A1) mAb IgG1 Isotype Control #5415, sections were incubated with GFAP (GA5) Mouse mAb (Alexa Fluor® 647 Conjugate) #3657 (red). Nuclei were labeled with Hoechst 33342 #4082 (blue).

Mouse Reactive Inflammasome Antibody Sampler Kit: Image 12

Western blot analysis of cell extracts and media from mouse bone marrow derived macrophages (mBMDM), untreated (-), or treated (+) with combinations of LPS #14011 (50 ng/ml, 4 hr) followed by nigericin (15 μM, 45 min) using Cleaved-IL-1β (Asp117) (D7V2A) Rabbit mAb (Mouse Specific) (upper) or total IL-1β (D3H1Z) Rabbit mAb (Mouse Specific) #12507 (lower).

Mouse Reactive Inflammasome Antibody Sampler Kit: Image 13

Western blot analysis of extracts from Raw264.7 cells, untreated (-) or treated with LPS (100 ng/mL, 6 hr; +), using IL-1β (D6D6T) Rabbit mAb (Mouse Specific) (upper), or β-Actin (D6A8) Rabbit mAb #8457 (lower).

Mouse Reactive Inflammasome Antibody Sampler Kit: Image 14

Western blot analysis of cell extracts from the cells or media from mouse bone marrow derived macrophages (mBMDM), untreated (-) or treated with Lipopolysaccharides (LPS) #14011 (50 ng/ml, 4 hr) followed by Nigericin (15 μM, 45 min) (+), using Cleaved Caspase-1 (Asp296) (E2G2I) Rabbit mAb (upper), or Caspase-1 (E2Z1C) Rabbit mAb (lower).

Mouse Reactive Inflammasome Antibody Sampler Kit: Image 15

Western blot analysis of extracts from various cell lines using Caspase-1 (E2Z1C) Rabbit mAb (upper), or β-Actin (D6A8) Rabbit mAb #8457 (lower).

Mouse Reactive Inflammasome Antibody Sampler Kit: Image 16

Western blot analysis of extracts from A20, J774A.1, and C2C12 cell lines using AIM2 Antibody (Mouse Specific) (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).

Mouse Reactive Inflammasome Antibody Sampler Kit: Image 17

Confocal immunofluorescent analysis of mouse primary bone marrow-derived macrophages (BMDMs) either untreated (upper left) or treated with LPS (50 ng/ml, 4 hr, middle) or LPS followed by ATP (5 mM, 45 min, upper right), and J774A.1 (lower left) or Raw 264.7 (lower right) cells, using ASC/TMS1 (D2W8U) Rabbit mAb (Mouse Specific) (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye). Note the translocation of ASC to inflammasomes following stimulation with LPS and ATP (white arrows).

Mouse Reactive Inflammasome Antibody Sampler Kit: Image 18

Western blot analysis of recombinant mouse IL-1β (mIL-1β) using IL-1β (D6D6T) Rabbit mAb (Mouse Specific).

Mouse Reactive Inflammasome Antibody Sampler Kit: Image 19

Western blot of A20, EL4, and M1 cell lines using Cleaved Caspase-1 (Asp296) (E2G2I) Rabbit mAb (upper), Caspase-1 (E2Z1C) Rabbit mAb (middle), or β-Actin (D6A8) Rabbit mAb #8457 (lower). The lack of staining in these cell lines using Cleaved Caspase-1 (Asp296) (E2G2I) Rabbit mAb demonstrates that it does not cross-react with full-length caspase-1.

Mouse Reactive Inflammasome Antibody Sampler Kit: Image 20

Western blot analysis of cell extracts from the cells or media from mouse bone marrow derived macrophages (mBMDM), untreated (-) or treated with Lipopolysaccharides (LPS) #14011 (50 ng/ml, 4hr) followed by Nigericin (15 μM, 45 min) (+) using Caspase-1 (E2Z1C) Rabbit mAb.

Mouse Reactive Inflammasome Antibody Sampler Kit: Image 21

Western blot analysis of extracts from J774A.1 cells, mock transfected (-) or transfected with mouse AIM2 siRNA (mAIM siRNA; +), using AIM2 Antibody (Mouse Specific) (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).

Mouse Reactive Inflammasome Antibody Sampler Kit: Image 22

Immunoprecipitation of ASC/TMS1 from J774A.1 cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is ASC (D2W8U) Rabbit mAb (Mouse Specific). Western blot analysis was performed using ASC/TMS1 (D2W8U) Rabbit mAb (Mouse Specific).

Mouse Reactive Inflammasome Antibody Sampler Kit: Image 23

Western blot analysis of extracts from J774A.1 and Raw 264.7 cells using ASC/TMS1 (D2W8U) Rabbit mAb (Mouse Specific) (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).

To Purchase # 20836T
Product # Size Price
20836T
1 Kit  (6 x 20 µl) N/A

Product Description

The Mouse Reactive Inflammasome Antibody Sampler Kit provides an economical means of detecting multiple inflammasome components. The kit includes enough antibodies to perform at least two western blot experiments with each primary antibody.

Specificity / Sensitivity

Each antibody in the Mouse Reactive Inflammasome Antibody Sampler Kit detects endogenous levels of its target protein. Caspase-1 (E2Z1C) Rabbit mAb detects endogenous levels of full-length mouse caspase-1; this antibody detects pro-caspase-1 and the p10 subunit of activated caspase-1. Cleaved Caspase-1 (Asp296) (E2G2I) Rabbit mAb detects endogenous levels of mouse caspase-1 only when cleaved at Asp296. A non-specific band is detected at 70 kDa in some cells. Cleaved-IL-1β (Asp117) (E7V2A) Rabbit mAb (Mouse Specific) recognizes endogenous levels of mouse IL-1β protein only when cleaved at Asp117.

Source / Purification

Monoclonal antibodies are produced by immunizing animals with recombinant mouse ASC/TMS1 protein, recombinant mouse IL-1β protein, or with synthetic peptides corresponding to residues surrounding Asp117 of mouse IL-1β, Asp296 of mouse caspase-1, residues near the carboxy terminus of mouse caspase-1, or residues surrounding Ala306 of mouse NLRP3. Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Val104 of mouse AIM2 protein. Polyclonal antibodies are purified by protein A and peptide affinity chromatography.

Background

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 (Reviewed 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/TMS1, 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.

Pathways & Proteins

Explore pathways + proteins related to this product.

Limited Uses

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