Cleaved-Caspase 8 (Asp384) Antibody - #AF5267

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Product: Cleaved-Caspase 8 (Asp384) Antibody
Catalog: AF5267
Description: Rabbit polyclonal antibody to Cleaved-Caspase 8 (Asp384)
Application: WB IHC IF/ICC
Reactivity: Human, Rat
Mol.Wt.: 45kDa(cleaved); 55kD(Calculated).
Uniprot: Q14790
RRID: AB_2837753

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MS Report
HPLC Report
MSDS
Data Sheet
COA
Protocols
WB Handbook
IHC Protocol
IF/ICC Protocol

Product Info

Source:
Rabbit
Application:
WB 1:500-1:2000, IF/ICC 1:100-1:500, IHC 1:50-1:200
*The optimal dilutions should be determined by the end user.
*Tips:

WB: For western blot detection of denatured protein samples. IHC: For immunohistochemical detection of paraffin sections (IHC-p) or frozen sections (IHC-f) of tissue samples. IF/ICC: For immunofluorescence detection of cell samples. ELISA(peptide): For ELISA detection of antigenic peptide.

Reactivity:
Human,Rat
Clonality:
Polyclonal
Specificity:
Cleaved-Caspase 8 (Asp384) Antibody detects endogenous levels of fragment of activated Caspase 8 resulting from cleavage adjacent to Asp384.
RRID:
AB_2837753
Cite Format: Affinity Biosciences Cat# AF5267, RRID:AB_2837753.
Conjugate:
Unconjugated.
Purification:
The antiserum was purified by peptide affinity chromatography using SulfoLink™ Coupling Resin (Thermo Fisher Scientific).
Storage:
Rabbit IgG in phosphate buffered saline , pH 7.4, 150mM NaCl, 0.02% sodium azide and 50% glycerol. Store at -20 °C. Stable for 12 months from date of receipt.
Alias:

Fold/Unfold

ALPS2B; Amyotrophic lateral sclerosis 2 chromosomal region candidate gene 12 protein; Apoptotic cysteine protease; Apoptotic protease Mch-5; Apoptotic protease Mch5; CAP4; CASP-8; CASP8; CASP8_HUMAN; Caspase 8; Caspase 8 apoptosis related cysteine peptidase; Caspase-8 subunit p10; CED 3; FADD Like ICE; FADD-homologous ICE/CED-3-like protease; FADD-like ICE; FLICE; FLJ17672; ICE-like apoptotic protease 5; MACH alpha 1/2/3 protein; MACH; MACH beta 1/2/3/4 protein; MCH5; MGC78473; MORT1 associated ced 3 homolog; MORT1-associated CED-3 homolog; OTTHUMP00000163717; OTTHUMP00000163720; OTTHUMP00000163724; OTTHUMP00000163725; OTTHUMP00000165062; OTTHUMP00000165063; OTTHUMP00000165064; OTTHUMP00000206552; OTTHUMP00000206582;

Immunogens

Immunogen:
Uniprot:

Q14790(CASP8_HUMAN) >>Visit HPA database.

Gene(ID):
CASP8(841)
Expression:
Q14790 CASP8_HUMAN:

Isoform 1, isoform 5 and isoform 7 are expressed in a wide variety of tissues. Highest expression in peripheral blood leukocytes, spleen, thymus and liver. Barely detectable in brain, testis and skeletal muscle.

Description:
Most upstream protease of the activation cascade of caspases responsible for the TNFRSF6/FAS mediated and TNFRSF1A induced cell death. Binding to the adapter molecule FADD recruits it to either receptor
Sequence:
MDFSRNLYDIGEQLDSEDLASLKFLSLDYIPQRKQEPIKDALMLFQRLQEKRMLEESNLSFLKELLFRINRLDLLITYLNTRKEEMERELQTPGRAQISAYRVMLYQISEEVSRSELRSFKFLLQEEISKCKLDDDMNLLDIFIEMEKRVILGEGKLDILKRVCAQINKSLLKIINDYEEFSKERSSSLEGSPDEFSNGEELCGVMTISDSPREQDSESQTLDKVYQMKSKPRGYCLIINNHNFAKAREKVPKLHSIRDRNGTHLDAGALTTTFEELHFEIKPHDDCTVEQIYEILKIYQLMDHSNMDCFICCILSHGDKGIIYGTDGQEAPIYELTSQFTGLKCPSLAGKPKVFFIQACQGDNYQKGIPVETDSEEQPYLEMDLSSPQTRYIPDEADFLLGMATVNNCVSYRNPAEGTWYIQSLCQSLRERCPRGDDILTILTEVNYEVSNKDDKKNMGKQMPQPTFTLRKKLVFPSD

PTMs - Q14790 As Substrate

Site PTM Type Enzyme
S16 Phosphorylation
K23 Ubiquitination
K34 Ubiquitination
K39 Ubiquitination
K63 Ubiquitination
S113 Phosphorylation
K121 Ubiquitination
K130 Ubiquitination
K148 Ubiquitination
K156 Acetylation
K156 Ubiquitination
K161 Ubiquitination
K169 Ubiquitination
K173 Ubiquitination
Y178 Phosphorylation
K183 Ubiquitination
S192 Phosphorylation
S209 Phosphorylation
S211 Phosphorylation
S219 Phosphorylation
K224 Ubiquitination
K229 Ubiquitination
Y235 Phosphorylation
K253 Ubiquitination
T263 Phosphorylation P51812 (RPS6KA3)
T273 Phosphorylation
S305 Phosphorylation
Y334 Phosphorylation
K344 Ubiquitination
S347 Phosphorylation Q16539 (MAPK14)
K351 Ubiquitination
K353 Ubiquitination
T373 Phosphorylation
S375 Phosphorylation
Y380 Phosphorylation P07948 (LYN) , P12931 (SRC)
S387 Phosphorylation P28482 (MAPK1) , P06493 (CDK1) , P27361 (MAPK3)
Y448 Phosphorylation P07948 (LYN)
K461 Ubiquitination
K473 Ubiquitination

Research Backgrounds

Function:

Most upstream protease of the activation cascade of caspases responsible for the TNFRSF6/FAS mediated and TNFRSF1A induced cell death. Binding to the adapter molecule FADD recruits it to either receptor. The resulting aggregate called death-inducing signaling complex (DISC) performs CASP8 proteolytic activation. The active dimeric enzyme is then liberated from the DISC and free to activate downstream apoptotic proteases. Proteolytic fragments of the N-terminal propeptide (termed CAP3, CAP5 and CAP6) are likely retained in the DISC. Cleaves and activates CASP3, CASP4, CASP6, CASP7, CASP9 and CASP10. May participate in the GZMB apoptotic pathways. Cleaves ADPRT. Hydrolyzes the small-molecule substrate, Ac-Asp-Glu-Val-Asp-|-AMC. Likely target for the cowpox virus CRMA death inhibitory protein. Isoform 5, isoform 6, isoform 7 and isoform 8 lack the catalytic site and may interfere with the pro-apoptotic activity of the complex. Cleaves RIPK1 at 'Asp-325' which is crucial for limiting apoptosis and necroptosis during embryonic development (By similarity).

PTMs:

Generation of the subunits requires association with the death-inducing signaling complex (DISC), whereas additional processing is likely due to the autocatalytic activity of the activated protease. GZMB and CASP10 can be involved in these processing events.

Phosphorylation on Ser-387 during mitosis by CDK1 inhibits activation by proteolysis and prevents apoptosis. This phosphorylation occurs in cancer cell lines, as well as in primary breast tissues and lymphocytes.

Subcellular Location:

Cytoplasm.

Extracellular region or secreted Cytosol Plasma membrane Cytoskeleton Lysosome Endosome Peroxisome ER Golgi apparatus Nucleus Mitochondrion Manual annotation Automatic computational assertionSubcellular location
Tissue Specificity:

Isoform 1, isoform 5 and isoform 7 are expressed in a wide variety of tissues. Highest expression in peripheral blood leukocytes, spleen, thymus and liver. Barely detectable in brain, testis and skeletal muscle.

Subunit Structure:

Heterotetramer that consists of two anti-parallel arranged heterodimers, each one formed by a 18 kDa (p18) and a 10 kDa (p10) subunit. Interacts with FADD, CFLAR and PEA15. Isoform 9 interacts at the endoplasmic reticulum with a complex containing BCAP31, BAP29, BCL2 and/or BCL2L1. Interacts with TNFAIP8L2 (By similarity). Interacts with CASP8AP2. Interacts with RFFL and RNF34; negatively regulate CASP8 through proteasomal degradation. Interacts with NOL3; decreases CASP8 activity in a mitochondria localization- and phosphorylation-dependent manner and this interaction is dissociated by calcium. Interacts with UBR2. Interacts with RIPK1 (By similarity). Interacts with stimulated TNFRSF10B; this interaction is followed by CASP8 proteolytic cleavage and activation.

(Microbial infection) Interacts with human cytomegalovirus/HHV-5 protein vICA/UL36; this interaction inhibits CASP8 activation.

(Microbial infection) Interacts with NleF from pathogenic E.coli.

(Microbial infection) Interacts with molluscum contagiosum virus protein MC160.

Family&Domains:

Isoform 9 contains a N-terminal extension that is required for interaction with the BCAP31 complex.

Belongs to the peptidase C14A family.

Research Fields

· Cellular Processes > Cell growth and death > p53 signaling pathway.   (View pathway)

· Cellular Processes > Cell growth and death > Apoptosis.   (View pathway)

· Cellular Processes > Cell growth and death > Apoptosis - multiple species.   (View pathway)

· Cellular Processes > Cell growth and death > Necroptosis.   (View pathway)

· Environmental Information Processing > Signal transduction > TNF signaling pathway.   (View pathway)

· Human Diseases > Drug resistance: Antineoplastic > Platinum drug resistance.

· Human Diseases > Endocrine and metabolic diseases > Non-alcoholic fatty liver disease (NAFLD).

· Human Diseases > Neurodegenerative diseases > Alzheimer's disease.

· Human Diseases > Neurodegenerative diseases > Huntington's disease.

· Human Diseases > Infectious diseases: Bacterial > Legionellosis.

· Human Diseases > Infectious diseases: Parasitic > Chagas disease (American trypanosomiasis).

· Human Diseases > Infectious diseases: Parasitic > Toxoplasmosis.

· Human Diseases > Infectious diseases: Bacterial > Tuberculosis.

· Human Diseases > Infectious diseases: Viral > Hepatitis B.

· Human Diseases > Infectious diseases: Viral > Human papillomavirus infection.

· Human Diseases > Infectious diseases: Viral > Herpes simplex infection.

· Human Diseases > Cancers: Overview > Pathways in cancer.   (View pathway)

· Human Diseases > Cancers: Overview > Viral carcinogenesis.

· Human Diseases > Cardiovascular diseases > Viral myocarditis.

· Organismal Systems > Immune system > Toll-like receptor signaling pathway.   (View pathway)

· Organismal Systems > Immune system > NOD-like receptor signaling pathway.   (View pathway)

· Organismal Systems > Immune system > RIG-I-like receptor signaling pathway.   (View pathway)

· Organismal Systems > Immune system > IL-17 signaling pathway.   (View pathway)

References

1). Bacillus spore-based oral carriers loading curcumin for the therapy of colon cancer. JOURNAL OF CONTROLLED RELEASE, 2018 (PubMed: 29274436) [IF=10.8]

Application: WB    Species: human    Sample: HT-29 cells

Figure.5 | Apoptosis detection of HT-29 colon cancer cells. (A) Apoptosis detection of HT-29 cells in different groups by flow cytometry, SFM without drug as control; (B) Apoptosis rates of HT-29 cells in different groups. (mean value ± SD, n=3, **p < 0.01, ***p < 0.001, compared with the control group); (C) Western blotting of the Bcl-2, p53, cleaved caspase-9, cleaved caspase-8,cleaved caspase-3; (D) Relative amount of these apoptosis-related proteins in different groups(mean value ± SD, n=3, *p < 0.05, **p < 0.01, ***p < 0.001, compared with the control group).
2). Long non-coding RNA RP11-197K6.1 as ceRNA promotes colorectal cancer progression via miR-135a-5p/DLX5 axis. Journal of translational medicine, 2024 (PubMed: 38760791) [IF=7.4]

Application: WB    Species: Human    Sample: HCT116 and SW480 cells

Fig. 2 Functional effects of lncRNA RP11-197K6.1 knockdown in CRC cells. A: Confirmation of lncRNA RP11-197K6.1 knockdown efficiency in HCT116 cells by RT-qPCR. B: Reduction in the proliferation of HCT116 and SW480 cells post-knockdown, as determined by proliferation assays. C: Increase in the apoptosis of HCT116 and SW480 cells following lncRNA RP11-197K6.1 knockdown, as measured by flow cytometry. D & E: Decrease in the migration and invasion of HCT116 and SW480 cells post-knockdown, as assessed by wound healing (scale = 200 μm) and Transwell assays (scale = 50 μm), respectively. 2 F: Changes in the levels of proteins related to the proliferation, apoptosis, and migration of HCT116 and SW480 cells after lncRNA RP11-197K6.1 knockdown, as analyzed by western blotting assay (**P 
3). Lysyl oxidase inhibits TNF-α induced rat nucleus pulposus cell apoptosis via regulating Fas/FasL pathway and the p53 pathways. LIFE SCIENCES, 2020 (PubMed: 32979358) [IF=6.1]

Application: WB    Species: rat    Sample: NP cells

Fig. 5. |LOX impaired apoptosis-related molecules expression in TNF-α-treated NP cells. A. Relative mRNA expressions of Bax, Bcl-2, caspase 8, cleaved caspase 9,cleaved caspase 3, Fas, FasL, and cleaved caspase 8 in rat NP cells with different treatments were analyzed by RT-qPCR. B. Protein expressions of Bax, Bcl-2, caspase 8, cleaved caspase 9 and cleaved caspase 3 in rat NP cells with different treatments were analyzed by western blot.
4). Carfilzomib suppressed LDHA-mediated metabolic reprogramming by targeting ATF3 in esophageal squamous cell carcinoma. Biochemical pharmacology, 2024 (PubMed: 38000560) [IF=5.8]
5). Akkermansia muciniphila Aspartic Protease Amuc_1434* Inhibits Human Colorectal Cancer LS174T Cell Viability via TRAIL-Mediated Apoptosis Pathway. INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, 2020 (PubMed: 32403433) [IF=5.6]

Application: WB    Species: human    Sample: LS174T cells

Figure 5. | Amuc_1434* mediated the activation of the apoptosis pathway in LS174T cells. (A) The expression of death receptor 4 (DR4), death receptor 5 (DR5), cysteinyl aspartate specific proteinase 8(caspase 8) and cysteinyl aspartate specific proteinase 3 (caspase 3) induced by Amuc_1434* in LS174T cells was dependent on tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). (a) LS174T cells were treated with 8 and 64 µg/mL Amuc_1434* for 24 h, respectively. The cell lysates were analyzed by Western blot.
6). Dehydrocorydaline attenuates myocardial ischaemia-reperfusion injury via the FoXO signalling pathway: a multimodal study based on network pharmacology, molecular docking, and experimental study. Journal of ethnopharmacology, 2024 (PubMed: 39222757) [IF=5.4]

Application: WB    Species: Rat    Sample: H9c2 cells

Fig. 7. Effects of DHC on the expression of predicted targets in the in vitro model of H/R injury. (A) Representative fluorescence images of the TUNEL assay. Photographs were taken at × 400 magnification. (B) Percentage of TUNEL-positive cells in each group. (C) Western blot showing the protein expression of cleaved-caspase 3 and cleaved-caspase 8 in each group. (D) Western blot showing the protein expression of p-FOXO1A, FOXO1A, CCND1, p-MDM2, and MDM2 in each group. (E) Relative protein levels of cleaved-caspase 3 and cleaved-caspase 8 measured in western blots (n = 3). (F) Relative protein levels of p-FOXO1A, FOXO1A, and p-FOXO1A/FOXO1A measured in western blots (n = 3). (G) Relative protein levels of p-MDM2, MDM2, and p-MDM2/MDM2 measured in western blots (n = 3). (H) Relative protein levels of CCND1 measured in western blots (n = 3). * indicates a significant difference compared to the control group; # indicates a significant difference compared to the H/R model group: *p < 0.05, **p < 0.01, #p < 0.05, ##p < 0.01. DHC: dehydrocorydaline, H/R: hypoxia/reoxygenation, TUNEL: TdT-mediated dUTP-biotin nick end labelling.
7). Antihepatoma peptide, scolopentide, derived from the centipede scolopendra subspinipes mutilans. World Journal of Gastroenterology, 2023 (PubMed: 37032730) [IF=4.3]

Application: WB    Species: Human    Sample: HepG2 cells

Figure 6 Antihepatoma mechanism of scolopentide. A: Flow cytometry suggested that apoptosis occurred in HepG2 cells after treatment with extracted scolopentide in vitro. B-I: Quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting showed that the expression of DR4 (B and C), DR5 (H and I), FADD (E and F), caspase-8 (F and G), and caspase-3 (C and D) was significantly upregulated in the scolopentide group. DR4 was the most considerably upregulated; I-K: qRT-PCR and western blotting showed that the expression of Cyto-C (I and J) and Bax/Bcl-2 (K) was not upregulated, which are key indicators of mitochondria dependence; L: Tumor ROS levels in the scolopentide group were higher than those in the vehicle group; M: c-FLIP, an inhibitory protein of caspase-8, was significantly downregulated in qRT-PCR; N: XIAP, an inhibitory protein of caspase-3, was insignificantly upregulated in qRT-PCR. DR4: Death receptor 4; DR5: Death receptor 5; FADD: Fas-associated death domain protein. n = 4 per group in qRT-PCR, n = 3 per group in Western blotting, n = 4 per group in ROS

Application: WB    Species: Human    Sample: HepG2 cells

Figure 6 Antihepatoma mechanism of scolopentide. A: Flow cytometry suggested that apoptosis occurred in HepG2 cells after treatment with extracted scolopentide in vitro. B-I: Quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting showed that the expression of DR4 (B and C), DR5 (H and I), FADD (E and F), caspase-8 (F and G), and caspase-3 (C and D) was significantly upregulated in the scolopentide group. DR4 was the most considerably upregulated; I-K: qRT-PCR and western blotting showed that the expression of Cyto-C (I and J) and Bax/Bcl-2 (K) was not upregulated, which are key indicators of mitochondria dependence; L: Tumor ROS levels in the scolopentide group were higher than those in the vehicle group; M: c-FLIP, an inhibitory protein of caspase-8, was significantly downregulated in qRT-PCR; N: XIAP, an inhibitory protein of caspase-3, was insignificantly upregulated in qRT-PCR. DR4: Death receptor 4; DR5: Death receptor 5; FADD: Fas-associated death domain protein. n = 4 per group in qRT-PCR, n = 3 per group in Western blotting, n = 4 per group in ROS
8). Chidamide, a subtype-selective histone deacetylase inhibitor, enhances Bortezomib effects in multiple myeloma therapy. Journal of Cancer, 2023 (PubMed: 34539893) [IF=3.9]

Application: WB    Species: Human    Sample: MM cells

Figure 4 A combination of Chidamide and Bortezomib increases production of ROS dependent DNA damage and the changes of cell apoptosis and cycle pathway in MM cells. (A) ARP-1 cells were pretreated with or without NAC (15.0 mmol/L) for 2 hours at 37°C and then incubated with CHI (1.0 µmol/L) and/or BTZ (5.0 nmol/L) for 24 hours, then ROS generation was detected. (B) ARP-1 cells were pretreated with or without 15 mmol/L NAC and then treated with Chidamide or Bortezomib alone or in combination, and cell viabilities were evaluated using CCK-8 assays. (C) The expression of γ-H2AX in ARP-1 cells treated with CHI (1.0 µmol/L) and/or BTZ (5.0 nmol/L) were determined by Western blot. (D) Representative images of γ-H2AX (Red) and nuclei (Blue) in ARP-1 cells treated with single agent or combination for 24 hours by immunofluorescence assay. Scale bars represent 20 µm. (E, F) Western blot analysis of the expressions of cleaved caspase3, cleaved caspase8, cleaved PARP-1 and HDAC1 in XG1 (E) and ARP-1 (F) cells after 48 hours treatment with single agent or in combination. Error bars indicate mean ± SD. **p < 0.01.
9). Noxa and Puma genes regulated by hTERT promoter can mitigate growth and induce apoptosis in hepatocellular carcinoma mouse model. Journal of Cancer, 2022 (PubMed: 35399714) [IF=3.9]

Application: WB    Species: human    Sample: HepG2 cells

Figure 4. |pcTERT-Noxa or pcTERT-Puma can trigger apoptosis in HepG2 cells by the mitochondrial and with death receptor pathways. (A) The expression levels of Bcl-2, Bax,cleaved Caspase-3, cleaved Caspase-9 and cleaved Caspase-8 were measured by western blot in HepG2 cells after transfection with pcTERT-Noxa or pcTERT-Puma for 72 h.
10). Exosomal miR-532–5p from bone marrow mesenchymal stem cells reduce intervertebral disc degeneration by targeting RASSF5. EXPERIMENTAL CELL RESEARCH, 2020 (PubMed: 32464126) [IF=3.7]

Application: WB    Species: Human    Sample: bone marrow mesenchymal stem cells (BMSCs)

Figure 4. Overexpression of miR-532-5p inhibited TNF-α-induced NPC apoptosis NPCs were transfected with miR-532-5p agomir, and then treated with TNF-α. (A) The relative expression of miR-532-5p was evaluated by qRT-PCR. (B) The apoptotic rate was determined by flow cytometry. (C) Representative images of TUNEL-positive cells by immunofluorescence staining.(D)The apoptotic proteins (cleaved-caspase-3, cleaved-caspase-9 and cleaved-capsase-8) were detected using Western blot. Each experiment was independently repeated at least three times.Data were shown as mean ± SD, * p<0.05, ** p<0.01*** p<0.001.
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