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98303
Mouse Reactive Pyroptosis Antibody Sampler Kit
Primary Antibodies
Antibody Sampler Kit

Mouse Reactive Pyroptosis Antibody Sampler Kit #98303

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Mouse Reactive Pyroptosis Antibody Sampler Kit: Image 1

Immunoprecipitation of Gasdermin D protein 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 Gasdermin D (E9S1X) Rabbit mAb. Western blot analysis was performed using Gasdermin D (E9S1X) Rabbit mAb. Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb (HRP Conjugate) #5127 was used as a secondary antibody.

Mouse Reactive Pyroptosis Antibody Sampler Kit: Image 2

Western blot analysis of extracts from mouse bone marrow derived macrophages (mBMDM), untreated (-) or treated with Lipopolysaccharides (LPS) #14011 (50 ng/ml, 4 hr; +) followed by Nigericin (sodium salt) #66419 (15 μM, indicated times; +), using Cleaved Gasdermin D (Asp276) (E3E3P) Rabbit mAb (upper) or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).

Mouse Reactive Pyroptosis Antibody Sampler Kit: Image 3

Western blot analysis of extracts from Raw 264.7 cells, untreated (-) or LPS-treated (1 μg/ml, overnight; +), using IL-1β (D3H1Z) Rabbit mAb (Mouse Specific).

Mouse Reactive Pyroptosis 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 Pyroptosis Antibody Sampler Kit: Image 5

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 Pyroptosis 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 Pyroptosis Antibody Sampler Kit: Image 7

Western blot analysis of extracts from Raw 264.7 or J774.1 cells, untreated (-) or treated with LPS (1 μg/ml, overnight; +) using Caspase-11 (17D9) Rat mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).

Mouse Reactive Pyroptosis Antibody Sampler Kit: Image 8

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 Pyroptosis Antibody Sampler Kit: Image 9

Immunohistochemical analysis of paraffin-embedded mouse lung using HMGB1 (D3E5) Rabbit mAb.

Mouse Reactive Pyroptosis Antibody Sampler Kit: 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.

Mouse Reactive Pyroptosis Antibody Sampler Kit: Image 11

Western blot analysis of extracts from control mouse bone marrow derived macrophages (mBMDM; lane 1) or mBMDM from Gasdermin D knockout mice (lane 2) using Gasdermin D (E9S1X) Rabbit mAb (upper) or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). The absence of signal in the Gasdermin D knockout cells confirms the specificity of the antibody for Gasdermin D. The mBMDM from Gasdermin D knockout mice were kindly provided by Dr. Douglas Golenbock, M.D., University of Massachusetts Medical School, Worcester, MA.

Mouse Reactive Pyroptosis Antibody Sampler Kit: Image 12

Western blot analysis of Mouse Interleukin-1β (mIL-1β) #5204 using IL-1β (D3H1Z) Rabbit mAb (Mouse Specific).

Mouse Reactive Pyroptosis Antibody Sampler Kit: Image 13

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 Pyroptosis Antibody Sampler Kit: Image 14

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 Pyroptosis Antibody Sampler Kit: Image 15

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 Pyroptosis Antibody Sampler Kit: Image 16

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 Pyroptosis Antibody Sampler Kit: Image 17

Immunohistochemical analysis of paraffin-embedded human colon carcioma using HMGB1 (D3E5) Rabbit mAb.

Mouse Reactive Pyroptosis Antibody Sampler Kit: Image 18

Western blot analysis of extracts from control PC-3 cells (lane 1) or Gasdermin D knockout PC-3 cells (lane 2) using Gasdermin D (E9S1X) Rabbit mAb (upper) or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). The absence of signal in the Gasdermin D knockout PC-3 cells confirms specificity of the antibody for Gasdermin D.

Mouse Reactive Pyroptosis Antibody Sampler Kit: Image 19

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 Pyroptosis Antibody Sampler Kit: Image 20

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 Pyroptosis Antibody Sampler Kit: Image 21

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 Pyroptosis Antibody Sampler Kit: Image 22

Immunohistochemical analysis of paraffin-embedded human lung carcioma using HMGB1 (D3E5) Rabbit mAb.

Mouse Reactive Pyroptosis Antibody Sampler Kit: Image 23

Western blot analysis of extracts from mouse bone marrow derived macrophages (mBMDM), untreated (-) or treated with Lipopolysaccharides (LPS) #14011 (50 ng/ml, 4 hr; +) followed by Nigericin (sodium salt) #66419 (15 μM, indicated times), using Gasdermin D (E9S1X) Rabbit mAb (upper), Cleaved Gasdermin D (Asp276) (E3E3P) Rabbit mAb #10137 (middle), or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).

Mouse Reactive Pyroptosis Antibody Sampler Kit: Image 24

Immunohistochemical analysis of paraffin-embedded J774A.1 cell pellet (left, positive) or RAW 264.7 cell pellet (right, negative) using ASC/TMS1 (D2W8U) Rabbit mAb (Mouse Specific).

Mouse Reactive Pyroptosis Antibody Sampler Kit: Image 25

Immunohistochemical analysis of paraffin-embedded human prostate carcioma using HMGB1 (D3E5) Rabbit mAb.

Mouse Reactive Pyroptosis Antibody Sampler Kit: Image 26

Western blot analysis of extracts from various cell lines using Gasdermin D (E9S1X) Rabbit mAb (upper) or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).

Mouse Reactive Pyroptosis Antibody Sampler Kit: Image 27

Immunohistochemical analysis of paraffin-embedded mouse forestomach using ASC/TMS1 (D2W8U) Rabbit mAb (Mouse Specific).

Mouse Reactive Pyroptosis Antibody Sampler Kit: Image 28

Western blot analysis of extracts from various cell lines using HMGB1 (D3E5) Rabbit mAb.

Mouse Reactive Pyroptosis Antibody Sampler Kit: Image 29

Western blot analysis of extracts from 293T cells, untransfected (-) or transfected with a construct expressing Myc/DDK-tagged full-length mouse Gasdermin D protein (mGSDMD-Myc/DDK; +), using Gasdermin D (E9S1X) Rabbit mAb (upper) or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).

Mouse Reactive Pyroptosis Antibody Sampler Kit: Image 30

Immunohistochemical analysis of paraffin-embedded mouse brain using ASC/TMS1 (D2W8U) Rabbit mAb (Mouse Specific).

Mouse Reactive Pyroptosis Antibody Sampler Kit: Image 31

Western blot analysis of extracts from THP-1 cells, differentiated with TPA (12-O-Tetradecanoylphorbol-13-Acetate) #4174 (50 ng/ml, overnight) and then treated with Lipopolysaccharides (LPS) #14011 (5 μg/ml, 6 hr), using Gasdermin D (E9S1X) Rabbit mAb (upper), Cleaved Gasdermin D (Asp275) (E7H9G) Rabbit mAb #36425 (middle), or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).

Mouse Reactive Pyroptosis Antibody Sampler Kit: Image 32

Immunohistochemical analysis of paraffin-embedded mouse colon using ASC/TMS1 (D2W8U) Rabbit mAb (Mouse Specific) (left) compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (right).

Mouse Reactive Pyroptosis Antibody Sampler Kit: Image 33

Immunohistochemical analysis of paraffin-embedded mouse thymus using ASC/TMS1 (D2W8U) Rabbit mAb (Mouse Specific).

Mouse Reactive Pyroptosis Antibody Sampler Kit: Image 34

Immunohistochemical analysis of paraffin-embedded mouse small intestine using ASC/TMS1 (D2W8U) Rabbit mAb (Mouse Specific).

Mouse Reactive Pyroptosis Antibody Sampler Kit: Image 35

Immunohistochemical analysis of paraffin-embedded Renca syngeneic tumor (top left), 4T1 syngeneic mammary tumor (top right), Renca cell pellet (bottom left), and 4T1 cell pellet (bottom right) using ASC/TMS1 (D2W8U) Rabbit mAb (Mouse Specific). Both tumors show staining of infiltrating immune cells. Note the presence of staining in the Renca tumor cells and the lack of staining in the 4T1 tumor cells consistent with staining results on corresponding cell pellets.

Mouse Reactive Pyroptosis Antibody Sampler Kit: Image 36

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 Pyroptosis Antibody Sampler Kit: Image 37

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 # 98303T
Product # Size Price
98303T
1 Kit  (9 x 20 µl) N/A

Product Includes Quantity Applications Reactivity MW(kDa) Isotype
Gasdermin D (E9S1X) Rabbit mAb 39754 20 µl
  • WB
  • IP
H M R 53, 30 Rabbit IgG
Cleaved Gasdermin D (Asp276) (E3E3P) Rabbit mAb 10137 20 µl
  • WB
M 31 Rabbit IgG
IL-1β (D3H1Z) Rabbit mAb (Mouse Specific) 12507 20 µl
  • WB
M 17,31 Rabbit IgG
Cleaved-IL-1β (Asp117) (E7V2A) Rabbit mAb (Mouse Specific) 63124 20 µl
  • WB
  • IP
M 17 Rabbit IgG
Caspase-1 (E2Z1C) Rabbit mAb 24232 20 µl
  • WB
  • IP
M 48, 10 Rabbit IgG
Cleaved Caspase-1 (Asp296) (E2G2I) Rabbit mAb 89332 20 µl
  • WB
  • IP
M 22 Rabbit IgG
Caspase-11 (17D9) Rat mAb 14340 20 µl
  • WB
  • IP
M 38, 43 Rat IgG2a
ASC/TMS1 (D2W8U) Rabbit mAb (Mouse Specific) 67824 20 µl
  • WB
  • IP
  • IHC
  • IF
  • F
M 22 Rabbit IgG
HMGB1 (D3E5) Rabbit mAb 6893 20 µl
  • WB
  • IHC
H M R Mk 29 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 

Product Description

The Mouse Reactive Pyroptosis Antibody Sampler Kit provides an economical means of detecting proteins that are used as readouts for pyroptosis. The kit includes enough antibodies to perform two western blot experiments with each primary antibody.

Specificity / Sensitivity

Gasdermin D (E9S1X) Rabbit mAb recognizes endogenous levels of total Gasdermin D protein. This antibody recognizes the 30 kDa amino terminal fragment produced during pyroptosis by caspase-1. Cleaved Gasdermin D (Asp276) (E3E3P) Rabbit mAb recognizes the amino terminal fragment of mouse Gasdermin D protein only when cleaved at Asp276. Caspase-1 (E2Z1C) Rabbit mAb detects endogenous levels of pro-caspase-1 and the p10 subunit of activated caspase-1. Cleaved Caspase-1 (Asp296) (E2G2I) Rabbit mAb detects endogenous levels of caspase-1 only when cleaved at Asp296. A non-specific band is detected at 70 kDa in some cells. IL-1β (D3H1Z) Rabbit mAb (Mouse Specific) recognizes endogenous levels of total mouse IL-1β protein. This antibody can detect 500 pg of mature recombinant mouse IL-1β. Cleaved IL-1β (Asp117) (E7V2A) Rabbit mAb (Mouse Specific) detects endogenous levels of mouse IL-1β protein only when cleaved at Asp117. Caspase-11 (17D9) Rat mAb recognizes endogenous levels of total caspase-11 protein. ASC/TMS1 (D2W8U) Rabbit mAb (Mouse Specific) recognizes endogenous levels of total mouse ASC/TMS1 protein. HMGB1 (D3E5) Rabbit mAb detects endogenous levels of total HMGB1. It does not cross-react with other HMGB proteins, including HMGB2 and HMGB3.

Source / Purification

Monoclonal antibodies are produced by immunizing animals with synthetic peptides corresponding to residues surrounding Leu60 of mouse Gasdermin D, His124 of mouse IL-1β, Ala137 of human HMGB1, a peptide near the carboxy terminus of mouse caspase-1, a recombinant fragment specific to the p30 subunit of mouse caspase-11, and a recombinant fragment specific to mouse ASC/TMS1 protein. Cleavage specific monoclonal antibodies are produced by immunizing animals with synthetic peptides corresponding to residues surrounding Asp296 of mouse caspase-1, Asp117 of mouse IL-1β, and Asp276 of mouse Gasdermin D protein.

Background

Pyroptosis is a regulated pathway of cell death with morphological features of necrosis, including cell swelling, plasma membrane pore formation, and engagement of an inflammatory response with the release of a number of damage-associated molecular patterns (DAMPs), such as HMGB1 and inflammatory cytokines like IL-1β and IL-18 (1,2). Pyroptosis is generally induced in cells of the innate immune system, such as monocytes, macrophages, and dendritic cells in the presence of pathogen-associated molecular patterns (PAMPs) expressed on microbial pathogens or by cell-derived DAMPs. It is induced through assembly of inflammasomes triggering proteolytic activation of caspase-1 which then cleaves inflammatory cytokines like IL-1β and IL-18 to their mature forms (3). A critical feature of pyroptosis is the cleavage of Gasdermin D by caspase-1 and mouse caspase-11 (or human caspase-4/5) (4-6). Upon cleavage, the N-terminal fragment of Gasdermin D oligomerizes to form a pore, allowing secretion of inflammatory DAMPs and cytokines. Canonical inflammasome assembly typically consists of a cytosolic-pattern recognition receptor (PPR; a nucleotide binding domain and leucine-rich repeat [NLR] or AIM2-like family members), an adaptor protein (ASC/TMS1), and pro-caspase-1. Distinct inflammasome complexes can recognize distinct PAMPs and DAMPs to trigger pyroptosis. The best characterized pathway triggered by the NLR, NLRP3, occurs through a two-step process. The first step is a priming signal, NF-κB is activated to induce the expression of a number of inflammasome components including NLRP3, pro-IL-1β, and pro-IL-18. In the second activation step, caspase-1 is activated and Gasdermin D and cytokines are proteolytically activated. In a non-canonical pathway, caspase-4 and caspase-5 can directly trigger Gasdermin D cleavage in monocytes following LPS stimulation (5,7).

  1. Frank, D. and Vince, J.E. (2019) Cell Death Differ 26, 99-114.
  2. Shi, J. et al. (2017) Trends Biochem Sci 42, 245-54.
  3. Malik, A. and Kanneganti, T.D. (2017) J Cell Sci 130, 3955-63.
  4. He, W.T. et al. (2015) Cell Res 25, 1285-98.
  5. Shi, J. et al. (2015) Nature 526, 660-5.
  6. Kayagaki, N. et al. (2015) Nature 526, 666-71.
  7. Viganò, E. et al. (2015) Nat Commun 6, 8761.

Pathways & Proteins

Explore pathways + proteins related to this product.

Limited Uses

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