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70934
Mouse Reactive PANoptosis Antibody Sampler Kit
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Mouse Reactive PANoptosis Antibody Sampler Kit #70934

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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 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).
Western blot analysis of extracts from Raw 264.7 cells, untreated (-) or LPS-treated (1 μg/ml, overnight; +), using IL-1β (D3H1Z) Rabbit mAb.
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 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).
Western blot analysis of extracts from various cell lines using MLKL (D6W1K) Rabbit mAb.
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.
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).
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 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).
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.
Western blot analysis of Mouse Interleukin-1β (mIL-1β) #5204 using IL-1β (D3H1Z) 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.
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).
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.
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.
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.
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).
Western blot analysis of extracts from various cell lines using RIP3 (D4G2A) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
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 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.
Western blot analysis of extracts from various cell lines using Gasdermin D (E9S1X) Rabbit mAb (upper) or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).
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.
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).
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).
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).
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.
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.
To Purchase # 70934T
Cat. # Size Price Inventory
70934T
1 Kit

Product Includes Quantity Applications Reactivity MW(kDa) Isotype
MLKL (D6W1K) Rabbit mAb 37705 20 µl
  • WB
  • IP
M 54 Rabbit IgG
Phospho-MLKL (Ser345) (D6E3G) Rabbit mAb 37333 20 µl
  • WB
  • IF
M 54 Rabbit IgG
Caspase-3 (D3R6Y) Rabbit mAb 14220 20 µl
  • WB
  • IP
H M R Mk 35, 19, 17 Rabbit IgG
Phospho-RIP3 (Thr231/Ser232) (E7S1R) Rabbit mAb 91702 20 µl
  • WB
  • IF
M 46-62 Rabbit IgG
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 12507 20 µl
  • WB
M 17,31 Rabbit IgG
Cleaved-IL-1β (Asp117) (E7V2A) Rabbit mAb 63124 20 µl
  • WB
  • IP
M 17 Rabbit IgG
RIP3 (D4G2A) Rabbit mAb 95702 20 µl
  • WB
  • IP
  • IF
  • F
M 46-62 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Rab Goat 

Product 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.

Specificity / Sensitivity

Each antibody in the Mouse Reactive PANoptosis Antibody Sampler Kit detects endogenous levels of its target protein. Phospho-MLKL (Ser345) (D6E3G) Rabbit mAb recognizes endogenous levels of mouse MLKL protein only when phosphorylated at Ser345. Weak, non-specific nuclear staining has been observed by immunofluorescence (IF-IC). Caspase-3 (D3R6Y) Rabbit mAb recognizes endogenous levels of total caspase-3 protein. This antibody detects full-length caspase-3 as well as the large subunit (p20) of caspase-3 resulting from cleavage during apoptosis. Phospho-RIP3 (Thr231/Ser232) (E7S1R) Rabbit mAb recognizes endogenous levels of RIP3 protein only when phosphorylated at Thr231/Ser232. This antibody may not recognize RIP3 when only singly phosphorylated at Thr231 or Ser232. 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 endogenous levels of the amino fragment of mouse Gasdermin D protein only when cleaved at Asp276. 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) recognizes endogenous levels of mouse IL-1β protein only when cleaved at Asp117.

Source / Purification

Monoclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of mouse MLKL, or synthetic peptides corresponding to residues surrounding Leu60 of mouse Gasdermin D, Asp276 of mouse Gasdermin D, His124 of mouse IL-1β, Asp117 of mouse IL-1β, Val370 of mouse RIP3, or synthetic phosphopeptides corresponding to residues surrounding Ser345 of mouse MLKL and Thr231/Ser232 of mouse RIP3, or recombinant protein specific to the p20 subunit of human caspase-3 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

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