Product: Cleaved-Caspase 9 (Asp353) Antibody
Catalog: AF5240
Source: Rabbit
Application: WB, IHC, IF/ICC, ELISA(peptide)
Reactivity: Human, Mouse, Rat
Prediction: Horse, Dog
Mol.Wt.: 17/38 kD(mature), 47kD(precursor); 46kD(Calculated).
Uniprot: P55211
RRID: AB_2837726

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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:
Horse(100%), Dog(100%)
Clonality:
Polyclonal
Specificity:
Cleaved-Caspase 9 (Asp353) Antibody detects endogenous levels of fragment of activated Caspase 9 resulting from cleavage adjacent to Asp353.
RRID:
AB_2837726
Cite Format: Affinity Biosciences Cat# AF5240, RRID:AB_2837726.
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:

A synthesized peptide derived from human Caspase 9 (Cleaved-Asp353).

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:
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. Proteolytically cleaves poly(ADP-ribose) polymerase (PARP).
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
100
Dog
100
Pig
71
Bovine
0
Sheep
0
Xenopus
0
Zebrafish
0
Chicken
0
Rabbit
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=19.160]

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=14.224]

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=14.224]

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). Ding Q et al. The role of the apoptosis-related protein BCL-B in the regulation of mitophagy in hepatic stellate cells during the regression of liver fibrosis. Exp Mol Med 2019 Jan 11;51(1):6 (PubMed: 30635551) [IF=12.153]

Application: WB    Species: mouse    Sample: HSC apoptosis

Fig. 3| Activation of mitophagy induces apoptosis in HSCs. a Mitochondrial DNA (mtDNA) measured by PicoGreen staining in LX2 cells; scale bar,10 μm. b Representative western blots of TOM20 with GAPDH serving as the internal reference. Bar graph represents the mean ± SEM. *P < 0.05 vs.the indicated groups. c TUNEL staining of LX2 cells from the indicated groups; scale bar, 25 μm. Bar graph represents the mean ± SEM. *P < 0.05 vs.the indicated groups. D Annexin V-FITC/PI double-staining and flow cytometry analysis of LX2 cells. Bar graph represents the mean ± SEM. **P < 0.01 vs. the indicated groups. e Representative western blots of cleaved caspase3, cleaved caspase9, collagen I and α-SMA. Bar graph represents the mean± SEM of three different experiments. *P < 0.05 and **P < 0.01 vs. the indicated groups

5). Yin L et al. Bacillus spore-based oral carriers loading curcumin for the therapy of colon cancer. J Control Release 2018 Feb 10;271:31-44 (PubMed: 29274436) [IF=11.467]

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).

6). 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=10.183]

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.

7). Wang S et al. Long noncoding RNAs regulated spermatogenesis in varicocele‐induced spermatogenic dysfunction. Cell Prolif 2022 Mar 17;e13220. (PubMed: 35297519) [IF=8.755]

Application: WB    Species: rat    Sample: Testicular

FIGURE 13| Validation of regulated signalling pathways, spermatogenic cell apoptosis and proliferation, and meiotic spermatocytes by Western blot. Representative Western blot images of PI3K, Akt, p-Akt, caspase-9, Bcl-2, Bax, PCNA, PLZF, REC8, STRA8, and SYCP3.

8). 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=7.033]

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.

9). Liu Z et al. Guizhi Fuling pill attenuates liver fibrosis in vitro and in vivo via inhibiting TGF-β1/Smad2/3 and activating IFN-γ/Smad7 signaling pathways. Bioengineered 2022 Apr;13(4):9357-9368. (PubMed: 35387552) [IF=6.832]

Application: WB    Species: human    Sample: LX-2 cells

Figure 1.| GZFL inhibits acetaldehyde-induced LX-2 cells activation through regulating cell viability and apoptosis.(f) Western blot was used to determine Bax, BCL-2, active caspase 3 and active caspase 9 expressions in LX-2 cells. **P < 0.01 vs. control group; ##P < 0.01 vs. AA group; n = 3.

10). Li X et al. Down-regulation of ROCK2 alleviates ethanol-induced cerebral nerve injury partly by the suppression of the NF-κB signaling pathway. Bioengineered 2020 Dec;11(1):779-790. (PubMed: 32684089) [IF=6.832]

Application: WB    Species: Rat    Sample: brain tissues

Figure 3. Down-regulation of ROCK2 protected hippocampal neurons against ethanol-induced cell death in rats. a. PI-stained neurons were photographed. DAPI (blue) was used to stain the nuclei, PI (red) was used to stained dead neurons. Scale bar = 40 μm.b. The expression of cleaved-caspase-3 and cleaved-caspase-9 in brain tissues of rats from each group was measured by Western blot. β-actin was used as a loading control.Data are expressed as mean± SD. ##p < 0.01 compared to Control group; **p < 0.01 compared to Ethanol+Lv-control group.

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