Total protein was extracted from each group as stated above and stored at −80 °C. Incubated with 1 μl anti-caspase-8 or ASC antibody overnight at 4 °C was 50 μl of protein. This reaction mixture was then incubated with protein A magnetic beads (2366538, Millipore, Temecula, CA, USA) for 30 min at 4 °C. Precipitates were washed three times with washing buffer and then eluted from protein A magnetic beads by boiling with 1 × SDS for 10 min at 90–100 °C. Western blot analysis was used to evaluate the expression of caspase-8 and ASC. Immunoprecipitation antibody, anti-caspase-8 (#8592, Cell Signaling Technology, Beverly, MA), anti-ASC (sc-22513,Santa Cruz, CA, USA); Western blot analysis anti-caspase-8 (sc-6134, Santa Cruz, CA, USA), anti-ASC (ab175449, Abcam), homophytic IgG as the negative control.
Anti caspase 8
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Anti-caspase-8 is a laboratory reagent used to detect and quantify the presence of caspase-8, an enzyme involved in the process of apoptosis or programmed cell death. This product can be used in various cell-based assays and research applications to study the role of caspase-8 in cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Anti β actin, by Santa Cruz Biotechnology (6 mentions)
Anti caspase 3, by Santa Cruz Biotechnology (5 mentions)
Anti caspase 3, by Cell Signaling Technology (5 mentions)
Anti parp 1, by Santa Cruz Biotechnology (4 mentions)
Anti caspase 8, by Cell Signaling Technology (3 mentions)
Anti cleaved caspase 3, by Santa Cruz Biotechnology (3 mentions)
Anti survivin, by Santa Cruz Biotechnology (3 mentions)
Anti lamin a c, by Abcam (3 mentions)
Lasmini 4000, by GE Healthcare (3 mentions)
Anti bcl 2, by Santa Cruz Biotechnology (3 mentions)
Anti bax, by Santa Cruz Biotechnology (3 mentions)
Anti caspase 9, by Santa Cruz Biotechnology (2 mentions)
Against β actin, by Santa Cruz Biotechnology (2 mentions)
Anti c myc, by Santa Cruz Biotechnology (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Anti β catenin, by BD (2 mentions)
Anti fasl, by Santa Cruz Biotechnology (2 mentions)
Anti bcl xl, by Santa Cruz Biotechnology (2 mentions)
Anti gapdh, by Cell Signaling Technology (2 mentions)
Anti caspase 9, by Cell Signaling Technology (2 mentions)
20 protocols using anti caspase 8
1
Immunoprecipitation and Western Blot Analysis of Caspase-8 and ASC
2
Comprehensive Cell Viability and Apoptosis Assay
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Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), and trypLE Express were purchased from Life Technologies (Carlsbad, CA, USA). CellTiter 96® AQueous One Solution was purchased from Promega (Madison, WI, USA). ELISA BrdU (colorimetric) was acquired from Roche (Basel, Switzerland). BD BioCoatTM MatrigelTM Invasion Chambers were acquired from Corning® (Corning, New York, NY, USA). The dead cell apoptosis kit with Annexin V-FITC/ PI was obtained from BD Bioscience (Franklin Lakes, NJ, USA). RNAse A and PI were purchased from Invitrogen (Carlsbad, CA, USA). Anti-caspase 8 and anti-caspase 9 antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). IRDye 800CW Goat anti-mouse IgG and IRDye 800CW Goat anti-rabbit IgG were acquired from LI-COR (Lincoln, NE, USA). Anti-caspase 3 was purchased from Abcam (Cambridge, UK). L-α-phosphatidylcholine (PC) and anti-vinculin antibody were obtained from Sigma-Aldrich (St. Louis, MI, USA). 1,2-Dioleoyl-sn-glycerol-3-phosphoserine sodium salt (Lipoid PS 18:1/18:1) was obtained from Lipoid GmbH (Ludwigshafen, Germany) and LabAssay Phospholipid was obtained from Wako (Osaka, Japan).
3
Western Blot Analysis of Apoptosis Markers
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Cells were harvested and lysed in SDS sample buffer (62.5 mM Tris-HCl, pH6.8, 10% glycerol, 2% SDS, 50 mM DTT and 0.01% bromphenol blue). Lysates were subjected to SDS-PAGE and transferred to PVDF membranes (Millipore). Membranes were blocked with 5% nonfat milk in Tris-buffered saline/0.1% Tween-20 and incubated at 4°C overnight with the following antibodies: anti-Axin 2, anti-Cyclin D1, anti-c-Myc, anti-Survivin, anti-caspase 3, anti-cleaved caspase 3, anti-caspase 8, anti-PARP-1 (Santa Cruz, CA), anti-Lamin A/C (Epitomics), anti-β-catenin (BD Biosciences), anti-phopho-β-catenin (Cell signaling Technology), and against β-actin (Santa Cruz, CA), followed by the corresponding horseradish peroxidase-conjugated secondary antibodies. Proteins of interest were visualized and imaged under chemi-luminescent detection using LASmini 4000 (GE Healthcare).
4
Analyzing Apoptosis Signaling Pathways
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Hoechst 33342, Protein A/G agarose and a NuPAGE system were purchased from Invitrogen Life Technologies (Grand Island, NY, USA). AbuRPFK(5-Fam)-NH2 peptide was obtained from Biomer Technologies (Pleasanton, CA, USA). Anti-cIAP1 antibody for western blot analysis was purchased from R&D Systems (Minneapolis, MN, USA). Anti-GAPDH, anti-caspase-8 and anti-PARP antibodies were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-RIPK1 antibody was obtained from BD Biosciences (Franklin Lakes, NJ, USA). Anti-TRAF2 antibody was obtained from Cell Signaling (Danvers, MA, USA). IAP antagonists were synthesized at TetraLogic Pharmaceuticals.
5
Mitochondrial Protein Isolation and Analysis
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Protein was isolated from homogenised samples and quantified using the BCA kit (Sigma-Aldrich, Huangpu, Shanghai, China). Ten percent SDS PAGE was done to separate the proteins and then transferred to a nitrocellulose membrane by means of electroblotting technique. Following this, the membrane was blocked using 5% blocking solution (non-fat milk). The membrane was kept in the blocking buffer containing primary antibodies overnight at 4°C (rabbit anti-cytochrome c 1 : 500, anti-smac 1 : 1000, anti-HIF-1α 1 : 500, anti-p53 1 : 1000, anti-FASL 1 : 300, anti-caspase-8 1 : 1000, anti-BID 1 : 500 and anti-β-actin 1 : 1000 (Santa Cruz Biotechnology, Chai Wan, Hong Kong, China). Next day, goat secondary antibody conjugated with horseradish peroxidase was added in the dilution 1 : 1000 in blocking buffer (non-fat milk). A chemiluminescence kit (Thermo Fisher Scientific, Pudong, Shanghai, China) was used to detect the proteins. The sample used was the mitochondrial fraction isolated from the rat spinal cord, as previously described [20 (link)].
6
Protein Expression Analysis of Cancer Markers
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Total protein was extracted from the samples, as previously described13 (link). Primary antibodies used were anti-B7-H3 (1:1000, Abcam, Cambridge, UK, ab 105922), anti-P16 (1:1000, Abcam, ab 108349), anti-P21 (1:1000, Abcam, ab 109199), anti-E2F (1:1000, ProteinTech, Chicago, USA, 12171-1-AP), anti-PARP-1 (1:200, Santa Cruz Biotech, CA, USA, sc-8007), anti-Caspase-8 (1:200, Santa Cruz Biotech, sc-56070), anti-Bax (1:200, Santa Cruz Biotech, sc-7480), anti-Bcl-2 (1:200, Santa Cruz Biotech, sc-7382), anti-Bcl-xl (1:200, Santa Cruz Biotech, sc-8392), anti-E7 (1:1000, Abcam, ab 30731), anti-Rb (Cell Signaling Technology, Danvers, MA, USA, #9309), and anti-GAPDH (1:5000, Cell Signaling Technology, #2118). Secondary antibodies were anti-mouse and anti-rabbit IgG peroxidase conjugates (1:5000, Merck Millipore, MA, USA). The ImageQuant LAS 4000 system (GE Healthcare Life Sciences, Logan, UT, USA) was used to detect these proteins, and the results were quantified by ImageJ software (NIH, Bethesda, MD, USA).
7
Apoptosis Pathway Protein Assays
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Z-VAD was purchased from Calbiochem. The antibodies used were as follows: anti-caspase-3 (Cell signaling, 9662), anti-caspase-8 (Cell signaling, 9746), anti-caspase-8 (Santa Cruz Biotech, SC-6136), anti-caspase-9 (Cell signaling, 9508), anti-TRADD (Cell signaling, 3684), anti-actin (Sigma, A2066), anti-Flag (Sigma, F-3165), anti-tubulin (Sigma, T7816), anti-PARP (Cell signaling, 9542), anti-Mcl1 (BD, 554103), anti-TL1A (Enzo, ALX-804-859-C100), and anti-TL1A (PEPROTECH, 500-P240). Recombinant proteins: FasL (R&D, 126-FL/CF), TNFα24 (link) and TRAIL25 (link). ELISA kits: TL1A (ENZO Life Science, APO-54N-027), TNF-alpha (RayBiotech, ELH-TNFα), FasL (RayBiotech, ELH-FASL), and TRAIL (RayBiotech, ELH-TRAIL).
8
Apoptotic Signaling Pathway Analysis
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Western Blot analysis was performed as described before35 (link). In brief, cells were homogenized in RIPA lysis buffer supplemented with protease inhibitor cocktail and phosphatase inhibitor cocktail (Sigma, St. Louis, MO). Protein lysates were resolved on SDS-PAGE gels before being transferred to the PVDF membrane. Anti-FADD, anti-Caspase-8, anti-RIP, and anti-RIP3 antibodies were purchased from Santa Cruz (sc-271748, sc-56070, sc-133102, and sc-374639 respectively). Mouse monoclonal anti-Actin antibody was used as a loading control. The densities of bands were quantitated using ImageJ software.
9
Apigenin-induced Apoptosis Pathway
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Apigenin (HPLC>98%) was purchased from the Nanjing TCM Institute of Chinese Materia Medica and was dissolved in sodium carbonate (20 mM). Purified mouse anti-Bax and anti-Bcl-2 antibodies were purchased from Biolegend (USA). Mouse monoclonal anti-phospho-ERK1/2, anti-caspase-3, anti-caspase-8, anti-phospho-p38, rabbit monoclonal anti-ERK1/2, anti-p38, mouse monoclonal anti-β-actin, rabbit/goat anti-mouse IgG and goat anti-rabbit IgG antibodies were purchased from Santa Cruz Biotechnology (USA). Mouse monoclonal anti-phospho-JNK and anti-JNK antibodies were purchased from Cell Signaling Technology (USA). Apoptosis, DNA Ladder Extraction Kit with Spin Column, DNA Molecular Weight Marker was purchased from Beyotime Institute of Biotechnology (Shanghai, China). In Situ Cell Death Kit, Fluorescein was purchased from Roche Applied Science, Mannheim, Germany.
10
Protein Extraction and Western Blot Analysis
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Tissue homogenates were resuspended in 1 × lysis buffer (1% NP-40, 100 mM Tris, pH 8.0, and 150 mM NaCl) containing a protease inhibitor mixture (Roche) and shaken 60 min at 4 °C. Samples were centrifuged and lysate was collected. Protein concentration was quantified using Pierce BCA protein assay kit (Thermo Fisher Scientific). Tissue lysates were mixed with 2 × Laemmli sample buffer and incubated at 100 °C for 5 min and equal amounts of protein were loaded into a 4–15% polyacrylamide gel for SDS/PAGE and transferred onto a nitrocellulose membrane (Bio-Rad). Membranes were incubated with the following primary antibodies: anti-caspase-1 (p20) (Casper-1, AdipoGen), anti-caspase-3 (#9662, Cell Signaling Technology), anti-caspase-7 (#9492, Cell Signaling Technology), anti-caspase-8 (#8592, Cell Signaling Technology), anti-caspase-8 (sc-81656, Santa Cruz Biotechnology), anti-Lipocalin-2 (AF1857-SP, R&D Systems) and anti-β-actin (sc-8432, Santa Cruz Biotechnology). Membranes were then probed with anti-mouse, anti-rabbit (Thermo Fisher Scientific) or anti-goat (Promega) secondary antibodies conjugated to horseradish peroxidase, and antibody complex were detected using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific).
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