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34886
Human Reactive PANoptosis Antibody Sampler Kit
Primary Antibodies
Antibody Sampler Kit

Human Reactive PANoptosis Antibody Sampler Kit #34886

Citations (0)
Simple Western™ analysis of lysates (0.1 mg/mL) from THP-1 cells treated with TPA (80nM, O/N) + LPS (1 µg/ml 15min) using IL-1β (D3U3E) Rabbit mAb #12703. 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 12-230 kDa separation module.
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.
Western blot analysis of extracts from various cell lines using RIP3 (E7A7F) XP® Rabbit mAb (upper) or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). Absence of signal in HeLa cells is predicted from published reports of epigenetic loss of expression and confirms the specificity of the antibody for RIP3.
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).
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.
Western blot analysis of extracts from various cell lines using MLKL (D2I6N) Rabbit mAb. KARPAS cell Line source: Dr. Abraham Karpas at the University of Cambridge.
Western blot analysis of extracts from THP-1 cells, differentiated with TPA #4174 (50 ng/ml, overnight) and then treated with LPS #14011 (5 μg/ml, indicated times), using Cleaved Gasdermin D (Asp275) (E7H9G) Rabbit mAb (upper), total Gasdermin D (L60) Antibody #93709 (middle), or β-Actin (D6A8) Rabbit mAb #8457 (lower).
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.
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.
Western blot analysis of recombinant Human Interleukin-1β (hIL-1β) #8900 using Cleaved-IL-1β (Asp116) (D3A3Z) Rabbit mAb.
Western blot analysis of HT-29 cells, untreated (-), or treated with combinations of the following treatments as indicated: Z-VAD (20 μM, added 30 min prior to other compounds; +), human TNF-α (hTNF-α, 20 ng/ml, 7 hr; +), SM-164 (100 nM, 7 hr; +), and necrostatin-1 (Nec-1, 50 μM, 7 hr; +), using Phospho-MLKL (Ser358) (D6H3V) Rabbit mAb (upper), or MLKL (D2I6N) Rabbit mAb #14993 (lower).
Western blot analysis of HT-29 cells, untreated (-) or treated with a combination of the following treatments as indicated: Z-VAD (20 μM, added 30 min prior to other compounds; +), Human Tumor Necrosis Factor-α #8902 (hTNF-α, 20 ng/ml, 7 hr; +), and SM-164 (100 nM, 7 hr; +), using Phospho-RIP3 (Ser227) (D6W2T) Rabbit mAb. To confirm phospho-specificity, membranes were either untreated (left) or treated with Calf Intestinal Phosphatase (CIP; right).
Immunoprecipitation of RIP3 protein from THP-1 cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is RIP3 (E7A7F) XP® Rabbit mAb. Western blot analysis was performed using RIP3 (E7A7F) XP® Rabbit mAb. Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb (HRP Conjugate) #5127 was used as a secondary antibody.
Western blot analysis of recombinant Human Interleukin-1β (hIL-1β) #8900 using IL-1β (D3U3E) Rabbit mAb.
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.
Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing Myc/DDK-tagged full-length human MLKL protein (hMLKL-Myc/DDK; +), using MLKL (D2I6N) Rabbit mAb (upper) and Myc-Tag (71D10) Rabbit mAb #2278 (lower).
Immunoprecipitation of Cleaved Gasdermin D (Asp725) from THP-1 cells differentiated with TPA #4174 (50 ng/ml, overnight) and then treated with LPS #14011 (5 μg/ml, 6 hr). Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is Cleaved Gasdermin D (Asp275) (E7H9G) Rabbit mAb. Western blot was performed using Cleaved Gasdermin D (Asp275) (E7H9G) Rabbit mAb. Anti-rabbit IgG, HRP-linked Antibody #7074 was used as a secondary antibody.
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.
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.
Western blot analysis of HT-29 cells, untreated (-) or treated with a combination of the following treatments as indicated: Z-VAD (20 μM, added 30 min prior to other compounds; +), Human Tumor Necrosis Factor-α #8902 (hTNF-α, 20 ng/ml, 7 hr; +), SM-164 (100 nM, 7 hr; +), and necrostatin-1 (Nec-1, 50 μM, 7 hr; +), using Phospho-RIP3 (Ser227) (D6W2T) Rabbit mAb (upper), RIP3 (E1Z1D) Rabbit mAb #13526 (middle), or β-Actin (D6A8) Rabbit mAb #8457 (lower).
Immunohistochemical analysis of paraffin-embedded human colon carcinoma (left), hepatocellular carcinoma (middle), or normal thymus (right) using RIP3 (E7A7F) XP® Rabbit mAb (top) or RIP3 Rabbit mAb (bottom). These two antibodies detect independent, unique epitopes on human RIP3 protein. The similar staining patterns obtained with both antibodies help to confirm the specificity of the staining.
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).
Immunohistochemical analysis of paraffin-embedded human ductal breast carcinoma using Cleaved Gasdermin D (Asp275) (E7H9G) Rabbit mAb.
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).
Western blot analysis of HT-29 cells or HT-29 RIPK1 KO cells, untreated (-) or treated with a combination of the following treatments as indicated: Z-VAD (20 μM, added 30 min prior to other compounds; +), Human Tumor Necrosis Factor-α #8902 (hTNF-α, 20 ng/ml, 7 hr; +), and SM-164 (100 nM, 7 hr; +), using Phospho-RIP3 (Ser227) (D6W2T) Rabbit mAb (upper), RIP3 (E1Z1D) Rabbit mAb #13526 (middle) or β-Actin (D6A8) Rabbit mAb #8457 (lower). HT-29 RIPK1 KO cells were kindly provided by Dr. Junying Yuan, Harvard Medical School, Boston, MA.
Immunohistochemical analysis of paraffin-embedded human ductal breast carcinoma using RIP3 (E7A7F) XP® Rabbit mAb.
Flow cytometric analysis of THP-1, untreated (blue, negative) or treated with LPS #14011 (100 ng/ml, 3 hr; green, positive) using IL-1β (D3U3E) Rabbit mAb (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.
Immunohistochemical analysis of paraffin-embedded human colon carcinoma using Cleaved Gasdermin D (Asp275) (E7H9G) Rabbit mAb in the presence of non-cleaved Gasdermin D peptide (left) or Asp275 cleavage-specific Gasdermin D peptide (right).
Western blot analysis of extracts from various cell lines using Gasdermin D (E9S1X) Rabbit mAb (upper) or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).
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).
Confocal immunofluorescent analysis of HT-29 cells, untreated (left), pre-treated with Z-VAD (20 μM, 30 min) followed by treatment with SM-164 (100 nM) and Human Tumor Necrosis Factor-α (hTNF-α) #8902 (20 ng/mL, 6 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-RIPK3 (Ser227) (D6W2T) Rabbit mAb (green). Actin filaments were labeled with DyLight 554 Phalloidin #13054 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
Immunohistochemical analysis of paraffin-embedded HT-29 cell pellet (left, high-expressing) or HeLa cell pellet (right, low-expressing) using RIP3 (E7A7F) XP® Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded human squamous cell lung carcinoma using Cleaved Gasdermin D (Asp275) (E7H9G) Rabbit mAb.
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).
Immunohistochemical analysis of paraffin-embedded normal human colon using RIP3 (E7A7F) XP® Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded human non-Hodgkin's Lymphoma using Cleaved Gasdermin D (Asp275) (E7H9G) Rabbit mAb.
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).
Immunohistochemical analysis of paraffin-embedded human colon carcinoma using RIP3 (E7A7F) XP® Rabbit mAb (left) compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (right).
Immunohistochemical analysis of paraffin-embedded human prostate carcinoma using Cleaved Gasdermin D (Asp275) (E7H9G) Rabbit mAb.
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.
Immunohistochemical analysis of paraffin-embedded human esophageal adenocarcinoma using RIP3 (E7A7F) XP® Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded human spleen (left, positive) and skeletal muscle (right, negative) using Cleaved Gasdermin D (Asp275) (E7H9G) Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded human squamous cell carcinoma of the tonsil using RIP3 (E7A7F) XP® Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded THP-1 cell pellets, differentiated with TPA #4174 (left) and then treated with LPS #14011 (right), using Cleaved Gasdermin D (Asp275) (E7H9G) Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded human endometrioid adenocarcinoma using RIP3 (E7A7F) XP® Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded human ulcerative colitis using Cleaved Gasdermin D (Asp275) (E7H9G) Rabbit mAb.
Confocal immunofluorescent analysis of HT-29 cells (left, positive) or HeLa cells (right, negative) using RIP3 (E7A7F) XP® Rabbit mAb (green). Actin filaments were labeled with DyLight 554 Phalloidin #13054 (red). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).
Flow cytometric analysis of SU-DHL-4 cells (blue, negative) and THP-1 cells (green, positive) using RIP3 (E7A7F) XP® Rabbit mAb (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.
To Purchase # 34886
Cat. # Size Qty. Price
34886T
1 Kit  (10 x 20 microliters)

Product Includes Quantity Applications Reactivity MW(kDa) Isotype
MLKL (D2I6N) Rabbit mAb 14993 20 µl
  • WB
H 54 Rabbit IgG
Phospho-MLKL (Ser358) (D6H3V) Rabbit mAb 91689 20 µl
  • WB
H 54 Rabbit IgG
Caspase-3 (D3R6Y) Rabbit mAb 14220 20 µl
  • WB
  • IP
H M R Mk 35, 19, 17 Rabbit IgG
RIP3 (E7A7F) XP® Rabbit mAb 10188 20 µl
  • WB
  • IP
  • IHC
  • IF
  • F
H 46-62 Rabbit IgG
Phospho-RIP3 (Ser227) (D6W2T) Rabbit mAb 93654 20 µl
  • WB
  • IF
H 46-62 Rabbit IgG
Cleaved Gasdermin D (Asp275) (E7H9G) Rabbit mAb 36425 20 µl
  • WB
  • IP
  • IHC
H 30 Rabbit IgG
Gasdermin D (E9S1X) Rabbit mAb 39754 20 µl
  • WB
  • IP
H M R 53, 30 Rabbit IgG
IL-1β (D3U3E) Rabbit mAb 12703 20 µl
  • WB
  • IF
  • F
H 17, 31 Rabbit IgG
Cleaved-IL-1β (Asp116) (D3A3Z) Rabbit mAb 83186 20 µl
  • WB
  • IF
H 17 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 

Product Description

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

Specificity / Sensitivity

Each antibody in the Human Reactive PANoptosis Antibody Sampler Kit detects endogenous levels of its target protein. MLKL (D2I6N) Rabbit mAb cross-reacts with an unidentified protein of 130 kDa in some cell lines. IL-1β (D3U3E) Rabbit mAb is not observed to detect endogenous levels of mature IL-1β. It can detect up to 100 pg of recombinant mature IL-1β. Caspase-3 (D3R6Y) Rabbit mAb detects full-length caspase-3 as well as the large subunit (p20) of caspase-3 resulting from cleavage during apoptosis. Gasdermin D (E9S1X) Rabbit mAb recognizes both the full-length Gasdermin D and the 30 kDa amino-terminal fragment produced during pyroptosis by caspase-1. Phospho-MLKL (Ser358) (D6H3V) Rabbit mAb recognizes endogenous levels of MLKL protein only when phosphorylated at Ser358. This antibody may also bind to MLKL when dually phosphorylated at Thr357 and Ser358. Phospho-RIP3 (Ser227) (D6W2T) Rabbit mAb recognizes endogenous levels of RIP3 protein only when phosphorylated at Ser227. A band is also detected at 30 kDa that appears to be a cleavage product of RIP3. Cleaved Gasdermin D (Asp275) (E7H9G) Rabbit mAb recognizes endogenous levels of Gasdermin D protein only when cleaved at Asp275. Cleaved-IL-1β (Asp116) (D3A3Z) Rabbit mAb recognizes endogenous levels of mature IL-1β protein only when cleaved at Asp116.

Source / Purification

Monoclonal antibodies are produced by immunizing animals with recombinant proteins specific to human IL-1β and the p20 subunit of human caspase-3; with synthetic peptides corresponding to residues near the carboxy termini of human MLKL and RIP3, or residues surrounding Leu60 of mouse Gasdermin D, Asp275 of human Gasdermin D, Asp116 of human IL-1β, Ser358 of human MLKL, and Ser227 of human RIP3 protein.

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).

Pathways

Explore pathways related to this product.

Limited Uses

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