Anti cleaved caspase 9

Manufactured by Cell Signaling Technology

Sourced in United States, China, United Kingdom

Anti-cleaved caspase-9 is a lab equipment product that detects the cleaved form of caspase-9, a key enzyme involved in the apoptosis (programmed cell death) pathway. It is used as a research tool to study apoptosis and cell signaling processes.

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Cleaved caspase-9 Anti-caspase-9 Caspase-9 Caspases

133 protocols using anti cleaved caspase 9

Western Blot Analysis of Apoptosis Markers

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The following antibodies were used in western blot: monoclonal rabbit anti-Smac (Epitomics), rabbit polyclonal anti-GAPDH (Abcam), rabbit polyclonal anti-cleaved PARP, anti-cleaved caspase-9, anti-caspase-3, anti-cleaved caspase-3 (Cell Signaling Technology), and rabbit polyclonal anti-VSV N (custom-made for the Luo lab). Details of antibody and caspase inhibitor usage are listed in Table 1.Table 1

Antibodies and Inhibitors

	Catalog No.	Vendor	Working Dilution
Inhibitors

Caspase-8 inhibitor (Z-IETD-FMK)	FMK007	R&D Systems	50 nM
Caspase-9 inhibitor (Z-LEHD-FMK)	FMK008	R&D Systems	50 nM

Reagents (Antibodies)

Anti-cIAP1	PA5-20066	Thermo Fisher	2 μg/mL
Anti-cIAP2	PA5-51700	Thermo Fisher	0.5 μg/mL
Anti-survivin	sc-17779	SCBT	1:300
anti-GAPDH	sc-32233	SCBT	1:1,000
anti-cleaved caspase-3	ab2302	Abcam	10 μg/mL
anti-cleaved PARP1 [4B5BD2]	ab110315	Abcam	1:1,000
Anti-VSV N	rabbit polyclonal	Luo lab	1:5,000

Li W., Turaga R.C., Li X., Sharma M., Enadi Z., Dunham Tompkins S.N., Hardy K.C., Mishra F., Tsao J., Liu Z.R., Fan D, & Luo M. (2019). Overexpression of Smac by an Armed Vesicular Stomatitis Virus Overcomes Tumor Resistance. Molecular Therapy Oncolytics, 14, 188-195.

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Generation and Characterization of Anti-BEX4 Antibodies

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Mouse polyclonal antibodies against human BEX4 protein were generated in C57BL/6 mice. Briefly, purified GST-BEX4 protein was injected four times intraperitoneally. Rabbit polyclonal antibodies against C-terminal polypeptides 89–106 of human BEX4 protein were commercially generated (Youngin Frontier, Seoul, Korea). The other antibodies used in this study were as follows: anti-GFP, anti-PLK1, anti-CDK1, anti-aurora A, anti-cIAP-1, anti-cdc20 (Santa Cruz Biotechnology, Dallas, TX, USA), anti-actin (Sigma-Aldrich, St. Louis, MO, USA), anti-poly(ADP-ribose) polymerase-1 (PARP), anti-cleaved-caspase 9, cleaved-caspase 7, anti-active-caspase 3, anti-phospho-threonine (p-Thr) (Cell Signaling Technology, Danvers, MA, USA), anti-aurora B (BD Biosciences PharMingen, San Diego, CA, USA), anti-securin (PTTG1; Zymed, San Francisco, CA, USA), anti-Myc (Bethyl Laboratories, Montgomery, TX, USA), and Alexa Fluor (Invitrogen, Leek, The Netherlands). The following reagents were used: MG132, cycloheximide (CHX), dimethyl sulfoxide (DMSO) (AG Scientific, San Diego, CA, USA), nocodazole (Sigma-Aldrich), and PLK1 kinase inhibitor BI2536 (Axon Medchem, Groningen, Netherlands).

Lee J.K., Ha G.H., Kim H.S, & Lee C.W. (2018). Oncogenic potential of BEX4 is conferred by Polo-like kinase 1-mediated phosphorylation. Experimental & Molecular Medicine, 50(10), 138.

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Western Blot Analysis of Apoptosis Markers

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All chemicals were sourced from Sigma-Aldrich (St. Louis, MO, USA), unless otherwise stated. For the western blot analysis, the following primary and secondary antibodies were used: Anti-cleaved caspase-6 (#9761), anti-cleaved caspase-7 (#9491), anti-cleaved caspase-8 (#9496), anti-cleaved caspase-9 (#9505), anti-PARP (#9542), anti-ATF6 (#65880), anti-BiP (#3177), anti-CHOP (#2895), anti-EGFR (#4267), and anti-MEK (#8727) from Cell Signaling Technology (Danvers, MA, USA). Anti-H-FABP (ab45966) and anti-CD36 (ab133625) from Abcam (Cambridge, UK), and anti-actin (clone AC-40).

Šrámek J., Němcová-Fürstová V., Polák J, & Kovář J. (2019). Hypoxia Modulates Effects of Fatty Acids on NES2Y Human Pancreatic β-cells. International Journal of Molecular Sciences, 20(14), 3441.

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Comprehensive Antibody Panel for Cell Signaling Analysis

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Catalog numbers and dilution fold are listed in parentheses. The following antibodies were purchased from Cell Signaling Technology: Anti-Bak (3792; 1:1000), anti-Bax (2772; 1:1000), anti-Bcl-2 (2870; 1:1000), anti-Bcl-xL (2764; 1:500), anti-CHK1 (2360; 1:1000), anti-phospho-CHK1 (S345; 2348; 1:1000), anti-cleaved caspase-3 (9661; 1:1000), anti-cleaved caspase-7 (9491; 1:1000), anti-cleaved caspase-8 (9496; 1:500), anti-cleaved caspase-9 (9501; 1:1000), anti-cytochrome c (4272; 1:500), anti-H2A.X (7631; 1:1000), anti-phospho-H2A.X (S139; 9718; 1:1000), anti-p53 (2524; 1:1000), anti-p53 (2527; 1:1000), anti-phospho-p53 (S6; 9285; 1:1000), anti-phospho-p53 (S15; 9284; 1:1000), anti-p21 (2946; 1:1000), and anti-voltage-dependent anion channel (VDAC; 4866; 1:1000). The following antibodies were purchased from Santa Cruz Biotechnology: Anti-CD95 (1023; 1:500), anti-cyclophilin 40, also known as anti-CYPD (137157; 1:400), anti-His tag (803; 1:500), anti-lamin A (20680; 1:1000), anti-MDM2 (965; 1:500), and anti-ubiquitin (8017; 1:200). Anti-PEPD (Ab86507; 1:500) and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; MAB374; 1:5000) were purchased from Abcam and EMD Millipore, respectively. Anti-mouse IgG-horseradish peroxidase (IgG-HRP; NA931V; 1:4000–5000) and anti-rabbit IgG-HRP (NA934V; 1:5000) were purchased from GE Healthcare.

Yang L., Li Y., Bhattacharya A, & Zhang Y. (2017). PEPD is a pivotal regulator of p53 tumor suppressor. Nature Communications, 8, 2052.

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Characterization of hnRNPK Methylation Mutants

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Wild-type (WT) pET23a-Trx-hnRNPK and pGEX4T-1-PRMT1 were prepared in a previous study (28 (link)). All mutant hnRNPK constructs were generated using a site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA). A total of nine arginine-to-lysine mutants were established: 2RK (R296K/R299K); 3RK-1 (R268K/R296K/R299K); 3RK-2 (R256K/R258K/R268K); 4RK-1 (R258K/R268K/R296K/R299K); 4RK-2 (R256K/R268K/R296K/R299K); 4RK-3 (R256K/R258K/R296K/R299K); 4RK-4 (R256K/R258K/R268K/R299K); 4RK-5 (R256K/R258K/R268K/R296K) and 5RK (R256K/R258K/R268K/R296K/R299K) and are listed in Supplementary Table S1. Full-length cDNAs encoding human PKCδ were constructed in the pCDNA4-myc/His vector. The recombinant catalytic fragment PKCδ was constructed in the pGEX4T1 vector. Pre-methylated hnRNPK was constructed in the pETDuet vector as described below.
The following antibodies were used in this study and purchased from vendors as indicated: anti-Myc tag (#115046) and GAPDH (#100118) (GeneTex lnc.); anti-hnRNPK/J (3C2) and anti-Flag (M2; Sigma-Aldrich); anti-mono and dimethyl arginine (AB412; Abcam); anti-Ser302-phosphorylation-hnRNPK (#18361; Santa Cruz Biotechnology); and anti-cleaved caspase3 (#9661), anti-cleaved caspase8 (#9496) and anti-cleaved caspase9 (#7237; Cell Signaling).

Yang J.H., Chiou Y.Y., Fu S.L., Shih I.Y., Weng T.H., Lin W.J, & Lin C.H. (2014). Arginine methylation of hnRNPK negatively modulates apoptosis upon DNA damage through local regulation of phosphorylation. Nucleic Acids Research, 42(15), 9908-9924.

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Protein Expression Analysis in Cells

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Total proteins were extracted from cells or tissues using RIPA Lysis Buffer. Protein concentration was measured by BCA Protein Assay Kit. To examine the expression of proteins, the same amount of total proteins was loaded on an 8-12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel. Proteins were transferred into PVDF membranes. After being blocked in skim milk solution, the membrane was incubated overnight separately with antibodies anti-β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-PI3K, anti-p-Akt, anti-p-Bad, anti-Cyt-c, anti-cleaved caspase 9, anti-cleaved caspase 3, anti-PARP, anti-DRP1, and anti-Mfn2 (Cell Signal, CST, USA). After that, the membrane was incubated with the secondary HRP-conjugated goat anti-rabbit antibodies (Santa Cruz Biotechnology). Proteins were visualized using an enhanced chemiluminescence kit from Thermo Fisher Scientific (Massachusetts, USA). ImageJ software (Alpha View SA) was used to perform densitometric analysis.

Song N., Jia L., Cao H., Ma Y., Chen N., Chen S., Lv X, & Yang G. (2020). Gypenoside Inhibits Endothelial Cell Apoptosis in Atherosclerosis by Modulating Mitochondria through PI3K/Akt/Bad Pathway. BioMed Research International, 2020, 2819658.

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Metformin and TRAIL-Induced Apoptosis

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Metformin was purchased from Wako (Richmond, VA, USA). TRAIL (Recombinant human) was purchased from Millipore (Millipore, Darmstadt, Germany.) Protein G PLUS-Agarose, Anti-Bax, anti-Bcl-2, anti-Mcl-1(IP), anti-Ub and anti-Bcl-xL were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-XIAP, anti-phospho ERK, anti-ERK, anti-phospho JAK2, anti-JAK2, anti-phospho AMPK, anti-AMPK, anti-phospho mTOR, anti-mTOR, anti-phospho AKT, anti-AKT, anti-phsopho GSK3β, anti-GSK3β, anti-Noxa, anti-Puma, anti-Bim, anti-phospho Mcl-1, anti-Mcl-1(WB), anti-cleaved caspase-3, anti-cleaved caspase-8, anti-cleaved caspase-9, anti-phospho STAT3, anti-STAT3, and anti-PARP-1 were purchased from Cell Signaling (Beverly, MA, USA). Anti-actin antibody was purchased from Sigma (Sigma, St. Louis, MO). Anti-Mule antibody was purchased from Abcam (Cat. No. ab70161). For the secondary antibodies, anti-mouse-IgG-HRP and anti-rabbit-IgG-HRP were purchased from Cell Signaling (Beverly, MA, USA).

Park S.H., Lee D.H., Kim J.L., Kim B.R., Na Y.J., Jo M.J., Jeong Y.A., Lee S.Y., Lee S.I., Lee Y.Y, & Oh S.C. (2016). Metformin enhances TRAIL-induced apoptosis by Mcl-1 degradation via Mule in colorectal cancer cells. Oncotarget, 7(37), 59503-59518.

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Protein Analysis of Heart Tissue Samples

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Heart tissues from study mice were homogenized and sonicated in a lysis buffer containing 20 mM Tris (pH 7.4), 150 mM NaCl, 1mM EDTA, 1 mM EGTA, 1% Triton, 0.1% sodium dodecyl sulfate, and a protease inhibitor cocktail. For in vitro study, isolated cardiomyocytes from WT mice treated with paraquat or rapamycin were sonicated in the lysis buffer as described above. Myocardial protein samples were incubated with anti-Akt, anti-phosphorylated Akt (Ser⁴⁷³), anti-Atg5, anti-LC3B, anti-Beclin1, anti-p38 MAPK, anti-phosphorylated p38 MAPK (Thr¹⁸⁰/Tyr¹⁸²), anti-Bad, anti-Bax, anti-Bcl2, anti-cleaved-caspase 3, anti-cleaved-caspase 9, anti-GRP78, anti-JNK, anti-phosphorylated JNK (Thr¹⁸³/Tyr¹⁸⁵), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; loading control) (1:1000; Cell Signaling Technology, Danvers, MA, USA), anti-p62 (1:1000; Guinea Pig; Enzo Life Sciences, Plymouth Meeting, PA, USA), anti-IRE1α, anti-phosphorylated IRE1α (Ser724) (1:1000, Abcam, Cambridge, MA, USA), anti-GADD153 (1:1000, Santa Cruz Biotechnology, Inc. Dallas, TX, USA) antibodies. Horseradish peroxidase-coupled secondary antibodies were used for membrane incubation. After immunoblotting, the films were scanned and detected with a Bio-Rad calibrated densitometer and the intensity of immunoblot bands was normalized with corresponding band intensity of GAPDH (37 (link)).

Wang Q, & Ren J. (2016). mTOR-Independent Autophagy Inducer Trehalose Rescues against Insulin Resistance-Induced Myocardial Contractile Anomalies: Role of p38 MAPK and Foxo1. Pharmacological research, 111, 357-373.

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Apoptosis Induction and NF-κB Modulation

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Dulbecco’s Modified Eagle Medium (DMEM) and Roswell Park Memorial Institute (RPMI)-1640 medium were purchased from Gibco (Shanghai, China). Fetal bovine serum (FBS) was obtained from Biological Industries (Cromwell, CT, USA). Acridine orange/ ethidium bromide (AO/EB) double fluorescence staining solution was obtained from Beyotime Institute of Biotechnology (Nanjing, China). Hoechst 33342 solution and cell counting kit-8 (CCK-8) were obtained from Dojindo Molecular Technologies, Inc. (Beijing, China). Pancaspase inhibitor (z-VAD-fmk) and NF-κB inhibitor (IKK 16) were bought from Selleckchem (Shanghai, China). Anti-Bcl-2, anti-Bax, anti-cytochrome c, anti-cleaved caspase-9 and anti-cleaved caspase-3 antibodies were acquired from Cell Signaling Technology (Shanghai, China). Anti-Ki-67, anti-PCNA, anti-E-cadherin, anti-N-cadherin, anti-Snail and anti-GAPDH antibodies were obtained from Abcam (Shanghai, China). Antibodies against NF-κB-p50, NF-κB-p65, IκB-α and p-IκB-α were obtained from Santa Cruz Biotechnology, Inc. (Dallas, Texas, USA).

Ji D., Zhong X., Huang P., Kang P., Leng K., Zheng W., Wang Z., Xu Y, & Cui Y. (2020). Deoxyelephantopin induces apoptosis via oxidative stress and enhances gemcitabine sensitivity in vitro and in vivo through targeting the NF-κB signaling pathway in pancreatic cancer. Aging (Albany NY), 12(11), 11116-11138.

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Apoptosis and EMT Regulation Assays

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Roswell Park Memorial Institute (RPMI)-1640 medium was bought from HyClone (Logan, UT, USA). Fetal bovine serum (FBS) was obtained from Thermo Scientific (Gibco, Shanghai, China). Cell counting kit-8 (CCK-8) was obtained from Dojindo Molecular Technologies (Tokyo, Japan). Dimethyl sulfoxide (DMSO) and Hoechst 33342 staining solution were purchased from Solarbio (Beijing, China). Primary antibodies against Bcl-2, Bax and cytochrome c were supplied by Proteintech (Rosemont, IL, USA). Anti-cleaved caspase-9 and anti-cleaved caspase-3 antibodies were purchased from Cell Signaling Technology (CST, Danvers, MA, USA). Antibodies against Ki-67, PCNA, Snail, E-cadherin, Vimentin and N-cadherin were acquired from Abcam (Cambridge, MA, USA).

Ji D., Zheng W., Huang P., Yao Y., Zhong X., Kang P., Wang Z., Shi G., Xu Y, & Cui Y. (2020). Huaier Restrains Cholangiocarcinoma Progression in vitro and in vivo Through Modulating lncRNA TP73-AS1 and Inducing Oxidative Stress. OncoTargets and therapy, 13, 7819-7837.

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