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).
Anti cleaved caspase 8
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-cleaved caspase-8 is a primary antibody that detects the cleaved form of caspase-8, a key enzyme involved in the initiation of apoptosis (programmed cell death). This antibody can be used to study the activation of the apoptotic pathway in various cell and tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti cleaved caspase 3, by Cell Signaling Technology (47 mentions)
Anti cleaved caspase 9, by Cell Signaling Technology (33 mentions)
Anti caspase 8, by Cell Signaling Technology (16 mentions)
Anti parp, by Cell Signaling Technology (13 mentions)
Anti bax, by Cell Signaling Technology (12 mentions)
Anti cleaved caspase 7, by Cell Signaling Technology (10 mentions)
Anti caspase 3, by Cell Signaling Technology (9 mentions)
Anti chop, by Cell Signaling Technology (8 mentions)
Anti bcl 2, by Cell Signaling Technology (8 mentions)
Anti caspase 9, by Cell Signaling Technology (8 mentions)
Anti cleaved parp, by Cell Signaling Technology (8 mentions)
Anti β actin, by Cell Signaling Technology (7 mentions)
Anti bcl xl, by Cell Signaling Technology (6 mentions)
Anti xiap, by Cell Signaling Technology (6 mentions)
Anti parp1, by Cell Signaling Technology (6 mentions)
Anti mcl 1, by Cell Signaling Technology (6 mentions)
Anti β actin, by Merck Group (6 mentions)
Bca kit, by Thermo Fisher Scientific (6 mentions)
Anti cleaved caspase 6, by Cell Signaling Technology (5 mentions)
Anti puma, by Cell Signaling Technology (5 mentions)
72 protocols using anti cleaved caspase 8
1
Western Blot Analysis of Apoptosis Markers
2
Molecular Mechanisms of Cell Death
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APAP and hydrogen peroxide were from Sigma (St. Louis, MO, USA). Z-VAD(OMe)-FMK (Z-VAD) and N-Acetyl-L-Carnosine (NAC) was from Cayman Chemical (Ann Arbor, MI, USA). Hi-Perfect transfection reagent was from Qiagen (Germanton, MD, USA). GsdmD siRNA and SCR control siRNA were from Origene (Rockville, MD, USA). Primary antibodies used were anti-cleaved caspase-3, anti-RIP, anti-phosphorylated RIP, anti-MLKL, anti-phosphorylated MLKL, anti-caspase-8, anti-cleaved caspase-8 from Cell Signaling Technologies (Danvers, MA, USA), and anti-gasdermin D from Santa Cruz Biotechnology (Dallas, TX, USA).
3
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.
4
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).
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).
5
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).
6
Multiparametric Tissue Analysis Protocol
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Histologic analysis was performed on snap frozen tissues that were fixed in acetone, blocked with 10% FCS, and stained with anti-active caspase-3 (BD Biosciences), anti-cleaved caspase-8 (Cell Signaling Technology), anti-PD-L1, anti-LY6C, anti-LY6G (all eBioscience), and anti-BAFF (R&D) antibodies. Fluorescent images were taken with an Axiocam 503 color microscope (ZEISS) and quantified using ImageJ. Conventional histology images were taken using the Brightfield microscope.
7
Immunoblotting of Apoptosis Markers
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Cells were lysed with cell lysis buffer (Cell Signaling Technology, Inc., Danvers, MA, USA) containing protease inhibitor cocktail (Sigma-Aldrich). Total cell lysates were subjected to SDS polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes by using an iBlot 2 Dry Blotting System (Thermo Fisher Scientific). Immunoblotting was carried out using the following antibodies; anti-caspase-3, anti-caspase-8, anti-cleaved caspase-8, anti-BIM, anti-Fas, and anti-β-actin antibodies (Cell Signaling Technology). Membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology), and imaging was performed with a ChemiDoc Touch imaging PC system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). All experiments were performed at least twice and the representative data of one experiment are shown.
8
Protein Expression Analysis via Western Blot
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Cells were lysed using a RIPA buffer, including a protease inhibitor cocktail (Thermo Scientific, USA). The proteins were separated by SDS-PAGE gels and transferred to PVDF membranes (Millipore, USA). After blocking with 5% non-fat milk, the membranes were incubated with primary antibodies at 4°C overnight. The membranes were washed three times and incubated for 2 hrs at room temperature with HRP-conjugated secondary antibodies. The antibodies used were the anti-E-cadherin (1:1,000, Cell Signaling), the anti-Vimentin (1:1,000, Cell Signaling), the anti-snail (1:1,000, Cell Signaling), the anti-PI3K p85 (1:1,000, Abacm), the anti-phospho-PI3K p85 (1:1,000, Cell Signaling), the anti-cleaved caspase-3 (1:1,000, Cell Signaling), the anti-cleaved caspase-8 (1:1,000, Cell Signaling), the anti-PARP (1:1,000, Abacm), the anti-total-AKT (1:1,000, Abacm), the anti-phospho-AKT (1:1,000, Cell Signaling), and anti-β-actin antibody (1:5,000, Santa cruz). Proteins were detected using a Bio-Rad ChemiDoc XRS+ System. Bio-Rad Image Lab software was used for densitometric analysis.
9
Oxidative Stress Biomarker Assay Protocol
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Griess reagent, 5,5’-Dithiobis (2-nitrobenzoic acid) and Lucigenin were purchased from Sigma-Aldrich. Hydrogen peroxide 33% was procured from PanReac AppliChem. The following antibodies were used in this work: Anti-fibronectin was obtained from GeneTex. Anti-MMP-9 and anti-Bcl2 were obtained from Proteintech. Anti-vimentin, anti-MMP-3, Anti-Akt, Anti-p38, Anti-Cleaved PARP1, Anti-p53 (acetyl K382), Anti-JNK, Anti-alpha smooth muscle Actin, Anti-NF-kB p65 (phospho S536), and anti-β catenin were all obtained from Abcam (Cambridge, UK). Anti-cleaved caspase-3, anti-cleaved aspase-9, and anti-cleaved caspase-8 were obtained from Cell Signaling. Rabbit Polyclonal JNK1/2/3 was obtained from ORIGENE. Anti-beta Actin was obtained from Thermo Fisher Scientific. Peroxidase AffiniPure Goat Anti-Rabbit IgG and Peroxidase AffiniPure Goat Anti-Mouse IgG were obtained from Jackson ImmunoResearch Inc. The following ELISA kits were used in this work: nitrotyrosine ELISA kit (Abcam, ab113848), Hydrogen Peroxide Colorimetric/Fluorometric assay kit (Biovision, K265-200), malondialdehyde assay kits (Northwest Life Sciences Specialties, NWK-MDA01) and Superoxide Dismutase assay kit (Cayman chemical, 706002).
10
Western Blot Analysis of Signaling Pathways
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DU145 cells were lysed in radioimmunoprecipitation assay buffer (150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 50 mM Tris–HCl [pH 7.5], 2 mM ethylenediaminetetraacetic acid) and 15 μg of protein was separated on 6–12% gels by sodium dodecyl sulfate-polyacrylamide gel electrophoresis Proteins were transferred to polyvinylidene difluoride membranes and then membranes were blocked in PBS with 0.1% Tween-20 containing 1% BSA and 1.5% skim milk for 1 h. After washing, the membranes were probed with primary antibody at 4 °C overnight, and then incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. The blot was developed using the EZ-western detection kit (Daeillab Service, Co., Seoul, Korea). Anti-cleaved caspase-8, −cleaved caspase-3, −PARP, −JNK, −p38, −p-ERK1/2, −p-SRC (Tyr-416 and Tyr-527), −SRC, −p-STAT3, −STAT3, −p-PI3K, −PI3K, −p-AKT (Ser-473), −AKT, −Ras, −p-Raf1 (Ser-259 and Ser-338), −p-MEK1/2, −p-p90RSK (Ser-380), and -RSK1/RSK2/RSK3 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-β-Actin, −p-JNK, −p-p38, −ERK2, and -Raf1 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz). The band intensities of specific antibodies were normalized and analyzed by ImageJ (Broken Symmetry Software, version 1.4.3.67).
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