Revision 1

#70934Store at -20C

1 Kit

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Cell Signaling Technology

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For Research Use Only. Not for Use in Diagnostic Procedures.
Product Includes Product # Quantity Mol. Wt Isotype/Source
MLKL (D6W1K) Rabbit mAb 37705 20 µl 54 kDa Rabbit IgG
Phospho-MLKL (Ser345) (D6E3G) Rabbit mAb 37333 20 µl 54 kDa Rabbit IgG
Caspase-3 (D3R6Y) Rabbit mAb 14220 20 µl 35, 19, 17 kDa Rabbit IgG
Phospho-RIP3 (Thr231/Ser232) (E7S1R) Rabbit mAb 91702 20 µl 46-62 kDa Rabbit IgG
Gasdermin D (E9S1X) Rabbit mAb 39754 20 µl 53, 30 kDa Rabbit IgG
Cleaved Gasdermin D (Asp276) (E3E3P) Rabbit mAb 10137 20 µl 31 kDa Rabbit IgG
IL-1β (D3H1Z) Rabbit mAb 12507 20 µl 17,31 kDa Rabbit IgG
Cleaved-IL-1β (Asp117) (E7V2A) Rabbit mAb 63124 20 µl 17 kDa Rabbit IgG
RIP3 (D4G2A) Rabbit mAb 95702 20 µl 46-62 kDa Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl Goat 

Please visit cellsignal.com for individual component applications, species cross-reactivity, dilutions, protocols, and additional product information.

Description

The Mouse Reactive PANoptosis Antibody Sampler Kit provides an economical means of detecting the activation of PANoptosis in mouse samples. The kit includes enough antibodies to perform two western blot experiments with each primary antibody.

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/mL BSA, 50% glycerol, and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibodies.

Background

Programmed cell death (PCD) plays important roles in organismal development and immune responses. There are three major PCD pathways: apoptosis, pyroptosis, and necroptosis. Apoptosis is a non-inflammatory cell death and is characterized by a series of proteolytic cleavage, beginning with the initiator caspases (caspases-8/9), then the executioner caspases (caspases-3/6/7), followed by cleavage of substrate proteins to drive apoptotic cell death (1,2). During pyroptosis, caspase-1 is proteolytically activated through a protein complex called inflammasome, then the activated caspase-1 can cleave Gasdermin D (GSDMD), IL-1β, and IL-18. The freed GSDMD N-terminal domains from the cleavage form pores in the plasma membrane to drive pyroptotic cell lysis and release of the cleaved and matured IL-1β and IL-18, as well as damage-associated molecular patterns (DAMPs) (3,4). The key steps in necroptosis include the receptor-interacting protein kinase 3 (RIPK3)-dependent phosphorylation of mixed lineage kinase domain-like protein (MLKL), translocation of phosphorylated MLKL to plasma membrane, and disruption of plasma membrane integrity (5,6). In contrast to the non-inflammatory nature of apoptosis, both pyroptosis and necroptosis are proinflammatory (7). While early studies of these PCD pathways focused on their distinct individual features and underlying mechanisms, recent findings point to crosstalk and redundancies among these processes under certain conditions, where the three pathways are activated, not independently of each other, and compensatory responses occur when one pathway is blocked. This new form of PCD with key features of pyroptosis, apoptosis, and/or necroptosis has been termed PANoptosis (8,9). PANoptosis is a coordinated cell death pathway driven by a cytoplasmic protein complex named the PANoptosome, whose components provide scaffold and catalytic functions to engage pyroptosis, apoptosis, and/or necroptosis (10,11).

  1. Hengartner, M.O. (2000) Nature 407, 770-6.
  2. Zimmermann, K.C. et al. (2001) Pharmacol Ther 92, 57-70.
  3. Shi, J. et al. (2017) Trends Biochem Sci 42, 245-254.
  4. Rathinam, V.A.K. and Chan, F.K. (2018) Trends Mol Med 24, 304-318.
  5. Christofferson, D.E. and Yuan, J. (2010) Curr Opin Cell Biol 22, 263-8.
  6. Vandenabeele, P. et al. (2010) Nat Rev Mol Cell Biol 11, 700-14.
  7. Nozaki, K. et al. (2022) Annu Rev Immunol 40, 469-498.
  8. Malireddi, R.K.S. et al. (2019) Front Cell Infect Microbiol 9, 406.
  9. Zheng, M. and Kanneganti, T.D. (2020) Immunol Rev 297, 26-38.
  10. Christgen, S. et al. (2020) Front Cell Infect Microbiol 10, 237.
  11. Samir, P. et al. (2020) Front Cell Infect Microbiol 10, 238.

Background References

    Trademarks and Patents

    Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
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    Orders: 877-616-CELL (2355)•[email protected]• Support: 877-678-TECH (8324)•[email protected]• Web: cellsignal.com
    For Research Use Only. Not for Use in Diagnostic Procedures.
    Revision 1
    #70934

    Mouse Reactive PANoptosis Antibody Sampler Kit

    Mouse Reactive PANoptosis Antibody Sampler Kit: Image 1 Expand Image
    Simple Western™ analysis of lysates (1 mg/mL) from Jurkat cells treated with Cytochrome C using Caspase-3 (D3R6Y) Rabbit mAb #14220. The virtual lane view (left) shows the target bands (as indicated) at 1:10 and 1:50 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:10 (blue line) and 1:50 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa separation module.
    Mouse Reactive PANoptosis Antibody Sampler Kit: Image 2 Expand Image
    Simple Western™ analysis of lysates (0.1 mg/ml) from J774A.1 cells RIP3 (D4G2A) Rabbit mAb #95702. The virtual lane view (left) shows the target band (as indicated) at 1:10 and 1:50 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:10 (blue line) and 1:50 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230kDa.
    Mouse Reactive PANoptosis Antibody Sampler Kit: Image 3 Expand Image
    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 PANoptosis Antibody Sampler Kit: Image 4 Expand Image
    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 Reactive PANoptosis Antibody Sampler Kit: Image 5 Expand Image
    Western blot analysis of various cell lines, untreated (-) or treated with Staurosporine #9953 (1 μM; 3 hr) or with Etoposide #2200 (25 μM, overnight), using Caspase-3 (D3R6Y) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower). MCF7 cells are negative for caspase-3 expression.
    Mouse Reactive PANoptosis Antibody Sampler Kit: Image 6 Expand Image
    Western blot analysis of L-929 cells, untreated (-), or treated with combinations of the following treatments as indicated: Z-VAD (20 μM, added 30 min prior to other compounds; +), Mouse Tumor Necrosis Factor-α (mTNF-α) #5178 (20 ng/ml, 4 hr; +), SM-164 (100 nM, 4 hr; +), and necrostatin-1 (Nec-1, 50 μM, 4 hr; +), using Phospho-MLKL (Ser345) (D6E3G) Rabbit mAb (upper), total MLKL (D6W1K) Rabbit mAb (Mouse Specific) #37705 (middle), or β-Actin (D6A8) Rabbit mAb #8457 (lower).
    Mouse Reactive PANoptosis Antibody Sampler Kit: Image 7 Expand Image
    Western blot analysis of extracts from various cell lines using MLKL (D6W1K) Rabbit mAb.
    Mouse Reactive PANoptosis Antibody Sampler Kit: Image 8 Expand Image
    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 PANoptosis Antibody Sampler Kit: Image 9 Expand Image
    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 (upper) or total IL-1β (E3H1Z) Rabbit mAb #12507 (lower).
    Mouse Reactive PANoptosis Antibody Sampler Kit: Image 10 Expand Image
    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 PANoptosis Antibody Sampler Kit: Image 11 Expand Image
    Western blot analysis of L-929 cells, untreated (-), or treated with combinations of the following treatments as indicated: Z-VAD (20 μM, added 30 min prior to other compounds; +), SM-164 (100 nM, 3 hr; +), and mouse TNF-α (20 ng/ml, 3 hr; +), using Phospho-RIP3 (Thr231/Ser232) (E7S1R) Rabbit mAb (upper), RIP3 (D8J3L) Rabbit mAb #15828 (middle), or β-Actin (D6A8) Rabbit mAb #8457 (lower).
    Mouse Reactive PANoptosis Antibody Sampler Kit: Image 12 Expand Image
    Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing full-length mouse RIP3 (mRIP3; +) using RIP3 (D4G2A) Rabbit mAb.
    Mouse Reactive PANoptosis Antibody Sampler Kit: Image 13 Expand Image
    Western blot analysis of Mouse Interleukin-1β (mIL-1β) #5204 using IL-1β (D3H1Z) Rabbit mAb.
    Mouse Reactive PANoptosis Antibody Sampler Kit: Image 14 Expand Image
    Western blot analysis of extracts from HCT116 cells (lane 1) or CASP3 knock-out cells (lane 2) using Caspase-3 (D3R6Y) Rabbit mAb #14220 (upper), and α-Actinin (D6F6) XP® Rabbit mAb #6487 (lower). The absence of signal in the CASP3 knock-out HCT116 cells confirms specificity of the antibody for CASP3.
    Mouse Reactive PANoptosis Antibody Sampler Kit: Image 15 Expand Image
    Confocal immunofluorescent analysis of L-929 cells, untreated (left), pre-treated with Z-VAD (20 μM, 30 min) followed by treatment with SM-164 (100 nM) and Mouse Tumor Necrosis Factor-α (mTNF-α) #5178 (20 ng/mL, 2.5 hr; center) and then post-processed with λ-phosphatase (right), using Phospho-MLKL (Ser345) (D6E3G) Rabbit mAb (green). Red = Propidium Iodide (PI)/RNase Staining Solution #4087 (fluorescent DNA dye).
    Mouse Reactive PANoptosis Antibody Sampler Kit: Image 16 Expand Image
    Immunoprecipitation of MLKL protein from BaF3 cells. Lane 1 is 10% input, lane 2 is immunoprecipitated with Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is MLKL (D6W1K) Rabbit mAb. Western blot was performed with MLKL (D6W1K) Rabbit mAb. A conformation specific secondary antibody was used to avoid reactivity with IgG.
    Mouse Reactive PANoptosis Antibody Sampler Kit: Image 17 Expand Image
    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 PANoptosis Antibody Sampler Kit: Image 18 Expand Image
    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. Western blot was performed using Cleaved-IL-1β (Asp117) (E7V2A) Rabbit mAb. Anti-Rabbit IgG, HRP-linked Antibody #7074 was used as a secondary antibody.
    Mouse Reactive PANoptosis Antibody Sampler Kit: Image 19 Expand Image
    Confocal immunofluorescent analysis of L-929 cells, untreated (left), pre-treated with Z-VAD (20 μM, 30 min) followed by treatment with SM-164 (100 nM) and Mouse Tumor Necrosis Factor-α (mTNF-α) #5178 (20 ng/mL, 2.25 hr; center), or pre-treated with Z-VAD followed by treatment with SM-164 and hTNF-α and post-processed with λ-phosphatase (right), using Phospho-RIP3 (Thr231/Ser232) (E7S1R) Rabbit mAb (green). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).
    Mouse Reactive PANoptosis Antibody Sampler Kit: Image 20 Expand Image
    Western blot analysis of extracts from various cell lines using RIP3 (D4G2A) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
    Mouse Reactive PANoptosis Antibody Sampler Kit: Image 21 Expand Image
    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 PANoptosis Antibody Sampler Kit: Image 22 Expand Image
    Western blot analysis of extracts from wild-type (+) or RIP3 knockout (-) mouse spleen using RIP3 (D4G2A) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower). Data were kindly provided by Dr. Junying Yuan, Harvard Medical School, Boston MA.
    Mouse Reactive PANoptosis Antibody Sampler Kit: Image 23 Expand Image
    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 PANoptosis Antibody Sampler Kit: Image 24 Expand Image
    Immunoprecipitation of RIP3 from L-929 cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is RIP3 (D4G2A) Rabbit mAb. Western blot analysis was performed using RIP3 (D4G2A) Rabbit mAb. A conformation-specific secondary antibody was used to avoid cross reactivity with IgG.
    Mouse Reactive PANoptosis Antibody Sampler Kit: Image 25 Expand Image
    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 PANoptosis Antibody Sampler Kit: Image 26 Expand Image
    Confocal immunofluorescent analysis of L-929 (left) and Neuro-2a (right) cells using RIP3 (D4G2A) Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
    Mouse Reactive PANoptosis Antibody Sampler Kit: Image 27 Expand Image
    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 PANoptosis Antibody Sampler Kit: Image 28 Expand Image
    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 PANoptosis Antibody Sampler Kit: Image 29 Expand Image
    Flow cytometric analysis of Neuro2A cells (blue) and L-929 cells (green) using RIPK3 mouse (D4G2A) Rabbit mAb (solid lines) compared to 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.
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    Orders: 877-616-CELL (2355)•[email protected]• Support: 877-678-TECH (8324)•[email protected]• Web: cellsignal.com
    For Research Use Only. Not for Use in Diagnostic Procedures.