Product: Caspase 9 Antibody
Catalog: AF6348
Source: Rabbit
Application: WB, IHC, IF/ICC, ELISA(peptide)
Reactivity: Human, Mouse, Rat
Prediction: Pig, Bovine, Horse, Sheep
Mol.Wt.: 46kD; 46kD(Calculated).
Uniprot: P55211
RRID: AB_2835042

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Product Info

Source:
Rabbit
Application:
WB 1:500-1:2000, IHC 1:50-1:200, IF/ICC 1:100-1:500, ELISA(peptide) 1:20000-1:40000
*The optimal dilutions should be determined by the end user.
Reactivity:
Human,Mouse,Rat
Prediction:
Pig(88%), Bovine(82%), Horse(91%), Sheep(90%)
Clonality:
Polyclonal
Specificity:
Caspase 9 Antibody detects endogenous levels of total Caspase 9.
RRID:
AB_2835042
Cite Format: Affinity Biosciences Cat# AF6348, RRID:AB_2835042.
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

APAF-3; APAF3; Apoptosis related cysteine peptidase; Apoptotic protease Mch-6; Apoptotic protease-activating factor 3; CASP-9; CASP9; CASP9_HUMAN; Caspase 9 apoptosis related cysteine peptidase; Caspase 9 Dominant Negative; Caspase 9c; Caspase-9; Caspase-9 subunit p10; ICE LAP6; ICE like apoptotic protease 6; ICE-LAP6; ICE-like apoptotic protease 6; MCH6; PPP1R56; protein phosphatase 1, regulatory subunit 56; RNCASP9;

Immunogens

Immunogen:
Uniprot:
Gene(ID):
Expression:
P55211 CASP9_HUMAN:

Ubiquitous, with highest expression in the heart, moderate expression in liver, skeletal muscle, and pancreas. Low levels in all other tissues. Within the heart, specifically expressed in myocytes.

Description:
This gene encodes a protein which is a member of the cysteine-aspartic acid protease (caspase) family. Sequential activation of caspases plays a central role in the execution-phase of cell apoptosis. Caspases exist as inactive proenzymes which undergo proteolytic processing at conserved aspartic residues to produce 2 subunits, large and small, that dimerize to form the active enzyme.
Sequence:
MDEADRRLLRRCRLRLVEELQVDQLWDALLSRELFRPHMIEDIQRAGSGSRRDQARQLIIDLETRGSQALPLFISCLEDTGQDMLASFLRTNRQAAKLSKPTLENLTPVVLRPEIRKPEVLRPETPRPVDIGSGGFGDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLELAQQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTS

Predictions

Predictions:

Score>80(red) has high confidence and is suggested to be used for WB detection. *The prediction model is mainly based on the alignment of immunogen sequences, the results are for reference only, not as the basis of quality assurance.

Species
Results
Score
Horse
91
Sheep
90
Pig
88
Bovine
82
Rabbit
78
Xenopus
71
Zebrafish
63
Dog
60
Chicken
0
Model Confidence:
High(score>80) Medium(80>score>50) Low(score<50) No confidence

PTMs - P55211 As Substrate

Site PTM Type Enzyme
K97 Ubiquitination
S99 Phosphorylation P17612 (PRKACA)
K100 Ubiquitination
T107 Phosphorylation
K117 Ubiquitination
T125 Phosphorylation P27361 (MAPK3) , P28482 (MAPK1) , Q13627 (DYRK1A) , P06493 (CDK1)
S133 Phosphorylation
S144 Phosphorylation Q05513 (PRKCZ)
Y153 Phosphorylation P00519 (ABL1) , A0A173G4P4 (Abl fusion)
S175 Phosphorylation
S183 Phosphorylation P17612 (PRKACA)
K189 Ubiquitination
S195 Phosphorylation P17612 (PRKACA)
S196 Phosphorylation Q9Y243 (AKT3) , P31751 (AKT2) , P31749 (AKT1) , Q13237 (PRKG2)
K204 Ubiquitination
T208 Phosphorylation
K210 Ubiquitination
K211 Ubiquitination
Y251 Phosphorylation
K278 Ubiquitination
T301 Phosphorylation
S302 Phosphorylation
S307 Phosphorylation
S310 Phosphorylation
K394 Ubiquitination
Y397 Phosphorylation

Research Backgrounds

Function:

Involved in the activation cascade of caspases responsible for apoptosis execution. Binding of caspase-9 to Apaf-1 leads to activation of the protease which then cleaves and activates caspase-3. Promotes DNA damage-induced apoptosis in a ABL1/c-Abl-dependent manner. Proteolytically cleaves poly(ADP-ribose) polymerase (PARP).

Isoform 2 lacks activity is an dominant-negative inhibitor of caspase-9.

PTMs:

Cleavages at Asp-315 by granzyme B and at Asp-330 by caspase-3 generate the two active subunits. Caspase-8 and -10 can also be involved in these processing events.

Phosphorylated at Thr-125 by MAPK1/ERK2. Phosphorylation at Thr-125 is sufficient to block caspase-9 processing and subsequent caspase-3 activation. Phosphorylation on Tyr-153 by ABL1/c-Abl; occurs in the response of cells to DNA damage.

Tissue Specificity:

Ubiquitous, with highest expression in the heart, moderate expression in liver, skeletal muscle, and pancreas. Low levels in all other tissues. Within the heart, specifically expressed in myocytes.

Subunit Structure:

Heterotetramer that consists of two anti-parallel arranged heterodimers, each one formed by a 35 kDa (p35) and a 10 kDa (p10) subunit. Caspase-9 and APAF1 bind to each other via their respective NH2-terminal CED-3 homologous domains in the presence of cytochrome C and ATP. Interacts (inactive form) with EFHD2. Interacts with HAX1. Interacts with BIRC2/c-IAP1, XIAP/BIRC4, BIRC5/survivin, BIRC6/bruce and BIRC7/livin. Interacts with ABL1 (via SH3 domain); the interaction is direct and increases in the response of cells to genotoxic stress and ABL1/c-Abl activation. Interacts with NleF from pathogenic E.coli.

Family&Domains:

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)

· Environmental Information Processing > Signal transduction > PI3K-Akt signaling pathway.   (View pathway)

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

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

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

· Human Diseases > Neurodegenerative diseases > Amyotrophic lateral sclerosis (ALS).

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

· Human Diseases > Infectious diseases: Bacterial > Legionellosis.

· Human Diseases > Infectious diseases: Parasitic > Toxoplasmosis.

· Human Diseases > Infectious diseases: Bacterial > Tuberculosis.

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

· Human Diseases > Infectious diseases: Viral > Influenza A.

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

· Human Diseases > Cancers: Specific types > Colorectal cancer.   (View pathway)

· Human Diseases > Cancers: Specific types > Pancreatic cancer.   (View pathway)

· Human Diseases > Cancers: Specific types > Endometrial cancer.   (View pathway)

· Human Diseases > Cancers: Specific types > Prostate cancer.   (View pathway)

· Human Diseases > Cancers: Specific types > Small cell lung cancer.   (View pathway)

· Human Diseases > Cancers: Specific types > Non-small cell lung cancer.   (View pathway)

· Human Diseases > Cardiovascular diseases > Viral myocarditis.

· Organismal Systems > Endocrine system > Thyroid hormone signaling pathway.   (View pathway)

References

1). Li X et al. Upregulation of BCL-2 by acridone derivative through gene promoter i-motif for alleviating liver damage of NAFLD/NASH. Nucleic Acids Res 2020 Jul 25;gkaa615. (PubMed: 32710621) [IF=11.501]

Application: WB    Species: human    Sample: HepG2

Figure 4. Effect of A22 on anti-apoptosis in 0.5 mM palmitic acid oil (PA) induced cell model. (A) Effect of A22 on cell viability for anti-apoptotic protective effect. (B) Effect of A22 on transcription of BCL-2 and BAX with measurement of mRNA levels. (C) Effect of A22 on protein expressions related with apoptosis (left), which were quantitatively analyzed (right). All the experiments were repeated for three times.

2). Yin Z et al. Formation of di-cysteine acrolein adduct decreases cytotoxicity of acrolein by ROS alleviation and apoptosis intervention. J Hazard Mater 2020 Apr 5;387:121686. (PubMed: 31780296) [IF=9.038]

3). Yin Z et al. Formation of di-cysteine acrolein adduct decreases cytotoxicity of acrolein by ROS alleviation and apoptosis intervention. J Hazard Mater 2020 Apr 5;387:121686. (PubMed: 31780296) [IF=9.038]

Application: WB    Species: Human    Sample: HBE (A) and Caco-2 (B) cells

Fig. 8 Effect of acrolein (ACR) or its adduct ACR-di-Cys with/without addition of 1mM cysteine on the cleavage of caspase-3, caspase-8 and PARP in HBE (A) and Caco-2 (B) cells. Left: western blot. Right: The data were expressed as relative intensity to GAPDH; Error bars represent standard deviation (n=3); Different letters indicate significant differences (p < 0.05) between treatments. 42 J

Application: WB    Species: Human    Sample: HBE and Caco-2 cells

Fig. 9 Effect of acrolein (ACR) or its adduct ACR-di-Cys with/without addition of 1mM cysteine on the expression of Bax, Bcl-2, cytochrome c and the cleavage of caspase-9 in HBE (A) and Caco-2 (B) cells. Left: western blot. Right: The data were expressed as relative intensity to GAPDH; Error bars represent standard deviation (n=3); Different letters indicate significant differences (p < 0.05) between treatments.

4). Wu H et al. LNC473 regulating APAF1 IRES-dependent translation via competitive sponging miR574 and miR15b: Implications in colorectal cancer. Mol Ther Nucleic Acids 2020 Sep 4;21:764-779. (PubMed: 32784109) [IF=7.032]

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

Figure 6. LNC473-miR574/miR15b-APAF1 Signaling Axis in HCT116 and SW480 Cells (A) IF and ISH combination assay revealing the co-localization of APAF1 protein with included ncRNAs in HCT116 cells. Scale bars, 5 mm. (B and C) The levels of APAF1 mRNA (B) and protein (C) were determined after interfering LNC473 expression in HCT116 and SW480 cells by qPCR and IF assays. Scale bars, 20 mm. (D) The expression of apoptosis-related proteins including APAF1 was detected after interfering LNC473 expression in HCT116 and SW480 cells by western blot assay. (E and F) Rescue experiments showing the APAF1 levels in HCT116 and SW480 cells with exposure to the co-transfection of pcDH-LNC473 vector and miR574-5p or miR15b-5p mimic by qPCR (E) and western blot (F) assays. (G) Pattern diagram of APAF1-CDS and APAF1-IRES-CDS vectors. (H and I) APAF1 protein expression was determined in HCT116 and SW480 cells treated with APAF1-IRES-CDS vector, or pcDH-LNC473 and APAF1-IRES-CD co-transfection by western blot (H) and IF assays (I). Scale bars, 5 mm. (J) The percentage (%) of cell apoptosis in cells upon co-overexpressing APAF1-CDS or APAF1-IRE-CDS and LNC473 as assayed by flow cytometry. All tests were performed at least three times. Data were expressed as mean ± SD. ns (nonsignificant), p > 0.05; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

5). Liu P et al. Cytotoxicity of adducts formed between quercetin and methylglyoxal in PC-12 cells. Food Chem 2021 Aug 1;352:129424. (PubMed: 33706136) [IF=6.306]

6). Liu Y et al. Increased autophagy in EOC re-ascites cells can inhibit cell death and promote drug resistance. Cell Death Dis 2018 Apr 1;9(4):419 (PubMed: 29549251) [IF=6.304]

Application: IHC    Species: human    Sample: EOC ascites cells

Fig. 2| Autophagy is increased and apoptosis is reduced in the EOC re-ascites group. a IHC analysis of LC-3, Belin-1, CASP-9, and c-CASP-3 expression in EOC ascites cells (scale bar =30µm). b, c Western blot analysis of autophagy/apoptosis-related proteins expressed in the chemosensitive group and re-ascites group (***p<0.001, **p<0.01, *p<0.05). d Immunofluorescence analysis of the chemosensitive group and re-ascites group with LC3 (green) and LAMP2 (red) staining. Nuclei were counterstained with DAPI (blue). e qRT-PCR analysis of the average relative mRNA expression levels of autophagy/apoptosis-related genes in the chemosensitive group and re-ascites group (**p<0.01, *p<0.05)

Application: IF/ICC    Species: human    Sample: ascites cells

Fig. 5 |Apoptosis is increased in the EOC chemosensitive group. a, b IHC and IF analysis of apoptotic protein expression in ovarian cancer samples. c, d Western blot analysis of apoptotic proteins expressed in the no-chemotherapy group and chemosensitive group (***p<0.001,**p<0.01, *p<0.05). e qRT-PCR analysis of the average relative mRNA expression levels of apoptosis-related genes in the no-chemotherapy group and chemosensitive group (*p<0.05)

Application: WB    Species: human    Sample: ascites cells

Fig. 5 |Apoptosis is increased in the EOC chemosensitive group. a, b IHC and IF analysis of apoptotic protein expression in ovarian cancer samples. c, d Western blot analysis of apoptotic proteins expressed in the no-chemotherapy group and chemosensitive group (***p<0.001,**p<0.01, *p<0.05). e qRT-PCR analysis of the average relative mRNA expression levels of apoptosis-related genes in the no-chemotherapy group and chemosensitive group (*p<0.05)

7). Liu Y et al. Increased autophagy in EOC re-ascites cells can inhibit cell death and promote drug resistance. Cell Death Dis 2018 Apr 1;9(4):419 (PubMed: 29549251) [IF=6.304]

8). He Y et al. Quercetin induces autophagy via FOXO1-dependent pathways and autophagy suppression enhances quercetin-induced apoptosis in PASMCs in hypoxia. Free Radic Biol Med 2017 Feb;103:165-176 (PubMed: 27979659) [IF=6.170]

Application: WB    Species: rat    Sample:


9). He Y et al. Quercetin induces autophagy via FOXO1-dependent pathways and autophagy suppression enhances quercetin-induced apoptosis in PASMCs in hypoxia. Free Radic Biol Med 2017 Feb;103:165-176 (PubMed: 27979659) [IF=6.170]

10). Wang L et al. Development of anisamide-targeted PEGylated gold nanorods to deliver epirubicin for chemo-photothermal therapy in tumor-bearing mice. Int J Nanomedicine 2019 Mar 8;14:1817-1833 (PubMed: 30880982) [IF=5.115]

Application: WB    Species:    Sample: PC-3 cells

Figure 6 |The expression of caspase 3, cleaved caspase 3 (phosphorylated form), caspase 9, cleaved caspase 9 (phosphorylated form), Bcl-2, Bax, and β-actin was determined using Western blotting.

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