Revision 1

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Orders:

877-616-CELL (2355)

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Support:

877-678-TECH (8324)

3 Trask Lane | Danvers | Massachusetts | 01923 | USA

For Research Use Only. Not for Use in Diagnostic Procedures.

Product Includes Product # Quantity Mol. Wt Isotype/Source
MLKL (D6W1K) Rabbit mAb3770520 µl54 kDaRabbit IgG
Phospho-MLKL (Ser345) (D6E3G) Rabbit mAb3733320 µl54 kDaRabbit IgG
Caspase-3 (D3R6Y) Rabbit mAb1422020 µl35, 19, 17 kDaRabbit IgG
Phospho-RIP3 (Thr231/Ser232) (E7S1R) Rabbit mAb9170220 µl46-62 kDaRabbit IgG
Gasdermin D (E9S1X) Rabbit mAb3975420 µl53, 30 kDaRabbit IgG
Cleaved Gasdermin D (Asp276) (E3E3P) Rabbit mAb1013720 µl31 kDaRabbit IgG
IL-1β (D3H1Z) Rabbit mAb1250720 µl17,31 kDaRabbit IgG
Cleaved-IL-1β (Asp117) (E7V2A) Rabbit mAb6312420 µl17 kDaRabbit IgG
RIP3 (D4G2A) Rabbit mAb9570220 µl46-62 kDaRabbit IgG
Anti-rabbit IgG, HRP-linked Antibody7074100 µlGoat

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).

Trademarks and Patents

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

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限制使用

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专品专有“专供研究使用”的专专或专似的专专声明, 且未专得美国食品和专品管理局或其他外国或国内专管机专专专任何用途的批准、准专或专可。客专不得将任何专品用于任何专断或治专目的, 或以任何不符合专专声明的方式使用专品。CST 专售或专可的专品提供专作专最专用专的客专,且专用于研专用途。将专品用于专断、专防或治专目的, 或专专售(专独或作专专成)或其他商专目的而专专专品,均需要 CST 的专独专可。客专:(a) 不得专独或与其他材料专合向任何第三方出售、专可、 出借、捐专或以其他方式专专或提供任何专品,或使用专品制造任何商专专品,(b) 不得复制、修改、逆向工程、反专专、 反专专专品或以其他方式专专专专专品的基专专专或技专,或使用专品开专任何与 CST 的专品或服专专争的专品或服专, (c) 不得更改或专除专品上的任何商专、商品名称、徽专、专利或版专声明或专专,(d) 只能根据 CST 的专品专售条款和任何适用文档使用专品 , (e) 专遵守客专与专品一起使用的任何第三方专品或服专的任何专可、服专条款或专似专专

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

Cell Signaling Technology Logo
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).
undefined Image 1: Mouse Reactive PANoptosis Antibody Sampler Kit
Western blot analysis of extracts from Raw 264.7 cells, untreated (-) or LPS-treated (1 μg/ml, overnight; +), using IL-1β (D3H1Z) Rabbit mAb.
undefined Image 2: Mouse Reactive PANoptosis Antibody Sampler Kit
Immunohistochemical analysis of paraffin-embedded Renca syngeneic tumor using IL-1β (D3H1Z) Rabbit mAb.
undefined Image 3: Mouse Reactive PANoptosis Antibody Sampler Kit
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

Cell Signaling Technology Logo
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.
undefined Image 4: Mouse Reactive PANoptosis Antibody Sampler Kit
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).
undefined Image 5: Mouse Reactive PANoptosis Antibody Sampler Kit
Western blot analysis of extracts from various cell lines using MLKL (D6W1K) Rabbit mAb.
undefined Image 6: Mouse Reactive PANoptosis Antibody Sampler Kit
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

Cell Signaling Technology Logo
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.
undefined Image 7: Mouse Reactive PANoptosis Antibody Sampler Kit
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).
undefined Image 8: Mouse Reactive PANoptosis Antibody Sampler Kit
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.
undefined Image 9: Mouse Reactive PANoptosis Antibody Sampler Kit
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

Cell Signaling Technology Logo
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).
undefined Image 10: Mouse Reactive PANoptosis Antibody Sampler Kit
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.
undefined Image 11: Mouse Reactive PANoptosis Antibody Sampler Kit
Western blot analysis of Mouse Interleukin-1β (mIL-1β) #5204 using IL-1β (D3H1Z) Rabbit mAb.
undefined Image 12: Mouse Reactive PANoptosis Antibody Sampler Kit
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

Cell Signaling Technology Logo
Immunohistochemical analysis of paraffin-embedded mouse spleen, untreated (left) or treated with Lipopolysaccharides (LPS) #14011 (right), using IL-1β (D3H1Z) Rabbit mAb.
undefined Image 13: Mouse Reactive PANoptosis Antibody Sampler Kit
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.
undefined Image 14: Mouse Reactive PANoptosis Antibody Sampler Kit
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.
undefined Image 15: Mouse Reactive PANoptosis Antibody Sampler Kit
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

Cell Signaling Technology Logo
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).
undefined Image 16: Mouse Reactive PANoptosis Antibody Sampler Kit
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.
undefined Image 17: Mouse Reactive PANoptosis Antibody Sampler Kit
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.
undefined Image 18: Mouse Reactive PANoptosis Antibody Sampler Kit
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

Cell Signaling Technology Logo
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.
undefined Image 19: Mouse Reactive PANoptosis Antibody Sampler Kit
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).
undefined Image 20: Mouse Reactive PANoptosis Antibody Sampler Kit
Western blot analysis of extracts from various cell lines using RIP3 (D4G2A) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
undefined Image 21: Mouse Reactive PANoptosis Antibody Sampler Kit
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

Cell Signaling Technology Logo
Immunohistochemical analysis of paraffin-embedded mouse lung, untreated (left) or treated with Lipopolysaccharides (LPS) #14011 (right), using IL-1β (D3H1Z) Rabbit mAb.
undefined Image 22: Mouse Reactive PANoptosis Antibody Sampler Kit
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).
undefined Image 23: Mouse Reactive PANoptosis Antibody Sampler Kit
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.
undefined Image 24: Mouse Reactive PANoptosis Antibody Sampler Kit
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

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Immunohistochemical analysis of paraffin-embedded CT26.WT syngeneic tumor using IL-1β (D3H1Z) Rabbit mAb (left) compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (right).
undefined Image 25: Mouse Reactive PANoptosis Antibody Sampler Kit
Western blot analysis of extracts from various cell lines using Gasdermin D (E9S1X) Rabbit mAb (upper) or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).
undefined Image 26: Mouse Reactive PANoptosis Antibody Sampler Kit
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.
undefined Image 27: Mouse Reactive PANoptosis Antibody Sampler Kit
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

Cell Signaling Technology Logo
Immunohistochemical analysis of paraffin-embedded RAW 264.7 cell pellets, untreated (left, negative) or treated with Lipopolysaccharides (LPS) #14011 (100 ng/ml, 7 hr), (positive, right), using IL-1β (D3H1Z) Rabbit mAb.
undefined Image 28: Mouse Reactive PANoptosis Antibody Sampler Kit
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).
undefined Image 29: Mouse Reactive PANoptosis Antibody Sampler Kit
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).
undefined Image 30: Mouse Reactive PANoptosis Antibody Sampler Kit
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

Cell Signaling Technology Logo
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).
undefined Image 31: Mouse Reactive PANoptosis Antibody Sampler Kit
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.
undefined Image 32: Mouse Reactive PANoptosis Antibody Sampler Kit
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.
undefined Image 33: Mouse Reactive PANoptosis Antibody Sampler Kit
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

Cell Signaling Technology Logo
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.
undefined Image 34: Mouse Reactive PANoptosis Antibody Sampler Kit
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.