Anti rip3

Manufactured by Abcam

Sourced in United States, United Kingdom

Anti-RIP3 is a protein that regulates a form of cell death known as necroptosis. It functions by binding to and inhibiting the activity of the RIP3 kinase, a key component of the necroptosis signaling pathway. Anti-RIP3 is used in research settings to study the role of necroptosis in various biological processes and disease states.

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Lab products found in correlation

Anti rip1, by BD (3 mentions) Anti caspase 8, by Cell Signaling Technology (2 mentions) Necrostatin 1, by Merck Group (2 mentions) Anti cleaved parp, by Cell Signaling Technology (1 mentions) Immobilon p, by Merck Group (1 mentions) Anti actin, by Merck Group (1 mentions) Anti bcl 2, by Cell Signaling Technology (1 mentions) Anti p62, by Merck Group (1 mentions) Western lightning, by PerkinElmer (1 mentions) Anti caspase3, by Novus Biologicals (1 mentions) Anti rip1, by Cell Signaling Technology (1 mentions) Anti ciap1, by Cell Signaling Technology (1 mentions) Anti atg5, by Santa Cruz Biotechnology (1 mentions) Anti lc3, by Merck Group (1 mentions) Protease and phosphatase inhibitor mini tablet, by Thermo Fisher Scientific (1 mentions) Anti β actin, by Abcam (1 mentions) Anti mlkl, by Abcam (1 mentions) Anti phospho mlkl, by Abcam (1 mentions) Bovine serum albumin (bsa), by Beyotime (1 mentions) Anti cleaved caspase 9, by Cell Signaling Technology (1 mentions)

Close spelling variants of this product

Anti-RIP3 antibody (5 citations) Anti-p-RIP3 (4 citations) Rabbit anti-RIP3 (4 citations) Mouse anti-p-RIP3 (2 citations) Anti-RIP3 polyclonal antibody (2 citations) Rabbit anti-RIP3 Ab (2 citations) Anti-RIP3 (phospho S227) antibody (2 citations) Rabbit anti-rat RIP3 polyclonal antibody (2 citations) Anti-RIP3/RIP3K (1 citations)

10 protocols using anti rip3

Immunoblot and Co-IP Protocol for Necroptosis

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Antibodies used for immunoblots were as follows: anti-ASS1 (Polaris, San Diego, CA, USA), anti-LC3 (Sigma), anti-p62 (Sigma), anti-RIP1 (Cell Signaling), anti-caspase 8 (Cell Signaling), anti-BCL-2 (Cell Signaling), anti-cIAP1 (Cell Signaling), anti-cleaved PARP (Cell Signaling), anti-caspase 3 (Imgenex, San Diego, CA, USA), anti-RIP3 (Abcam, Cambridge, MA, USA), anti-Atg5 (Santa Cruz, Dallas, TX, USA), anti-Atg7 (Microbiology Laboratories, Woburn, MA, USA), and anti-actin (Sigma). Cells were lysed in RIPA buffer and protein concentrations were determined by BCA kit (Pierce, Waltham, MA, USA). In all, 25–40 μg of proteins was resolved by NuPAGE (Invitrogen) and transferred onto PVDF membranes (Immobilon-P, Millipore, Darmstadt, Germany). Antibody detection was accomplished using enhanced chemiluminescence (Western Lightning, PerkinElmer, Melville, NY, USA).
For co-IP, SK-LMS-1 cells were treated with PBS, 1 μg/ml ADI-PEG20, 20 μM chloroquine, or both for 3 days. Following treatment, cells were lysed in 0.2% NP-40 buffer and protein concentration was determined by BCA kit (Pierce). Lysates were incubated with anti-RIP1 (Cell Signaling) and protein A/G beads (Pierce). The immunoprecipitates were subsequently immunoblotted with anti-RIP3 (Abcam), anti-RIP1 (Cell Signaling), anti-caspase 8 (Cell Signaling) and anti-actin (Sigma) as described above.

Bean G.R., Kremer J.C., Prudner B.C., Schenone A.D., Yao J.C., Schultze M.B., Chen D.Y., Tanas M.R., Adkins D.R., Bomalaski J., Rubin B.P., Michel L.S, & Van Tine B.A. (2016). A metabolic synthetic lethal strategy with arginine deprivation and chloroquine leads to cell death in ASS1-deficient sarcomas. Cell Death & Disease, 7(10), e2406-.

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Western Blot Analysis of Cell Death Signaling

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Retina tissue and cells were harvested and lysed in RIPA buffer containing 10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% sodium dodecyl sulfate (SDS), 1% Nonidet P-40, and 1% sodium deoxycholate, and was supplemented with protease and phosphatase inhibitor mini tablets (Thermo Fisher Scientific, Waltham, MA). The protein concentration was determined with bicinchoninic acid protein assay. Western blotting was performed as previously described [23 (link)]. Briefly, 30 μg of protein was loaded per lane on a 10% SDS-PAGE. The membrane blots were saturated with 5% BSA in PBST for 1 h at room temperature and then incubated overnight at 4 °C with antibodies. Primary antibodies used included anti-RIP1 (BD Bio-Sciences, 1:1000), anti-RIP3 (Abcam, 1:1000), anti-phospho-RIP1 (Invitrogen, 1:1000), anti-phospho-RIP3 (Abcam, 1:1000), anti-MLKL (Abcam, 1:1000), anti-phospho-MLKL (Abcam, 1:1000), and anti-β-actin (Abcam, 1:2000) antibodies. Band intensities were measured using the Image J software (US National Institutes of Health). Co-immunoprecipitation assays were performed using the Thermo Scientific Pierce co-IP kit according to the manufacturer’s instruction. The levels of TNF-α and MCP-1 in cultured microglia was detected with ELISA kits (R&D Systems, Minneapolis, MN) according to manufacturer’s instruction.

Huang Z., Liang J., Chen S., Ng T.K., Brelén M.E., Liu Q., Yang R., Xie B., Ke S., Chen W, & Huang D. (2023). RIP3-mediated microglial necroptosis promotes neuroinflammation and neurodegeneration in the early stages of diabetic retinopathy. Cell Death & Disease, 14(3), 227.

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Immunofluorescence Analysis of RIP1 and RIP3 Expression

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Immunofluorescence was used to detect RIP1 and RIP3 expression. Cells were treated with TNF-α (40 µM) and Nec-1 (50 µM), washed with PBS, fixed with 4% paraformaldehyde for 15 min at 4°C, and were then permeabilized with 0.5% Triton X-100 for 10 min at room temperature. After blocking with 5% BSA (Beyotime Institute of Biotechnology) for 1 h at 37°C, the cells were incubated with either anti-RIP1 (cat. no. 3493; 1:200; Cell Signaling Technology, Inc.) or anti-RIP3 (cat. no. ab62344; 1:200; Abcam) antibodies overnight at 4°C. The slides were then incubated with Alexa Fluor^® 488-conjugated goat anti-rabbit IgG antibody (cat. no. ab150077; 1:200; Abcam,) for 1 h at 37°C. The nuclei were then stained with DAPI for 5 min at room temperature. Images were obtained under a fluorescence microscope.

Lin B., Jin Z., Chen X., Zhao L., Weng C., Chen B., Tang Y, & Lin L. (2020). Necrostatin-1 protects mice from acute lung injury by suppressing necroptosis and reactive oxygen species. Molecular Medicine Reports, 21(5), 2171-2181.

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Apoptosis and Oxidative Stress Assay

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Bicinchoninic acid (BCA), trichloroacetic acid (TCA), sulforhodamine B (SRB), propidium iodide, RNase A, dimethyl sulfoxide (DMSO) and catalase were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA). Roswell Park Memorial Institute (RPMI) 1640 medium, Dulbecco Modified Eagle Medium (DMEM)/high glucose, Minimum Essential Media (MEM), fetal bovine serum (FBS), trypsin-EDTA solution (1X), antibiotic-antimycotic solution (100X), and PBS (1X) were purchased from HyClone Laboratories, Inc. (South Logan, UT, USA). Anti-caspase-9, anti-cleaved caspase-9, anti-caspase-3, anti-BID, anti-p-p53 (Ser15), anti-Prx1, anti-caspase-8, and anti-cleaved caspase-8 were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-cleaved caspase-3, anti-Bax, anti-Bcl-2, anti-p53, anti-catalase, anti-SOD-1, anti-SOD-2, anti-GPx-1, anti-Trx, anti-PrxI/II, anti-β-actin, and all secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-BIM, anti-PARP, anti-cleaved PARP, and fluorescein isothiocyanate (FITC) Annexin V apoptosis detection kit I were purchased from BD Biosciences (CA, USA). Anti-RIP-3 was purchased from Abcam (Cambridge, MA, USA). 2′,7′-dichlorodihydrofluorescein diacetate (DCFH₂-DA) was purchased from Invitrogen (Carlsbad, CA, USA).

Kang J.I., Hong J.Y., Choi J.S, & Lee S.K. (2016). Columbianadin Inhibits Cell Proliferation by Inducing Apoptosis and Necroptosis in HCT116 Colon Cancer Cells. Biomolecules & Therapeutics, 24(3), 320-327.

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Investigating CVA21-Induced Cell Death

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Cells were infected with CVA21 MOIs at IC₅₀, which for T24 was 11.4, 1.0 for TCCSUP, and 1.8 for 5637. Cell viability was determined 72 hr post-infection by MTS assay (Promega), and the IC₅₀ value was calculated using CalcuSyn software (Biosoft, UK).
Apoptotic cell death was determined with the annexin V-PE kit (BD Biosciences). FACS analysis was performed as above.
Caspase Glo 3/7 assay (Promega) was used to evaluate caspase 3 and 7 activity post-CVA21 infection. T24, TCCSUP, and 5637 cells were plated in 96-well plates and incubated overnight. Cells were treated with 0.5 μg/mL Mitomycin-C and CVA21 as above. At 24, 48, and 72 hr post-treatment, Caspase Glo reagent was used as per manufacturer instructions and luminescence measured using a Varioskan Flash (Thermo Scientific) plate reader.
To confirm the death pathways involved, the pan-caspase inhibitor zVAD and the inhibitor of necroptosis, Necrostatin-1 (Sigma-Aldrich), were used. Necrostatin-1 (30 μM) was added 45 min prior to Mitomycin-C and/or virus addition, whereas the zVAD (20 mM) was added after Mitomycin-C and/or virus treatment. Cell viability was measured using MTS assay (Promega).
In addition, whole-cell lysates were prepared, followed by immunoblotting with anti-human PARP (no. 9542; Cell Signaling Technology, USA), anti-RIP1 (BD Biosciences), and anti-RIP-3 (Abcam) antibodies.

Annels N.E., Arif M., Simpson G.R., Denyer M., Moller-Levet C., Mansfield D., Butler R., Shafren D., Au G., Knowles M., Harrington K., Vile R., Melcher A, & Pandha H. (2018). Oncolytic Immunotherapy for Bladder Cancer Using Coxsackie A21 Virus. Molecular Therapy Oncolytics, 9, 1-12.

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Apoptosis and Necroptosis Pathway Analysis

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Anti-cleaved caspase-3 (#9664), anti-β-actin (#13E5) bodies were purchased from Cell Signaling Technology, Inc. (Cell Signaling, MA). Anti-RIP 3 (#ab56164) bodies were purchased from Abcam, Inc. (Abcam, Cambridge, UK). Monoclonal anti-actin (α-sarcomeric) antibody (#A2172), necrostatin-1 (#N9037), Hoechst 33258 (#861405) and 2′, 7′-Dichlorofluorescin diacetate (DCFH-DA; #D6883) were purchased from Sigma-Aldrich Co (St. Louis, MO). In situ cell death detection kit (#11684795910) was purchased from Roche Ltd (Roche, USA). Superoxide dismutase (SOD; #A001-1), malonaldehyde (MDA; #A002-1), and catalase (CAT; #A007-1) detection kit were purchased from JIANCHENG Bioengineering Institute (Nanjing City, P.R China). Cellular glutathione peroxidase (GPx; #S0056) assay kit and caspase inhibitor Z-VAD-FMK (#C1202) were purchased from Beyotime Institute of Biotechnology (Nantong City, P.R China).

Gao X., Zhang H., Zhuang W., Yuan G., Sun T., Jiang X., Zhou Z., Yuan H., Zhang Z, & Dong H. (2014). PEDF and PEDF-derived peptide 44mer protect cardiomyocytes against hypoxia-induced apoptosis and necroptosis via anti-oxidative effect. Scientific Reports, 4, 5637.

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Necroptosis Detection in Osteocytes

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The presence/absence of RIP1 and RIP3 expression in Dex(+)/hypoxia(+) was investigated to confirm whether the conditions required for the development of necroptosis in osteocytic cells in an H-D stress environment were satisfied. Cultured cells were fixed in 4% paraformaldehyde, washed in phosphate buffered saline (PBS), and permeabilized with 0.3% Triton X-100 in PBS. Nonspecific binding was blocked by incubating sections with 10% bovine serum albumin (Dako Cytomation, Santa Clara, CA, USA) in PBS for 15 min. They were incubated with anti-RIP1 (Bioss, MA, USA) and anti-RIP3 (abcam, Cambridge, UK) antibody for 2 h at concentrations of 20.0 or 5.0 µg/mL, followed by a fluorescent-labeled secondary antibody (Alexa 594, or 488, Thermo Fisher Scientific, Waltham, MA, USA) and by DAPI for 30 min. After washing, a prolong diamond antifade mountant (Thermo Fisher Scientific) was added, and cover slips were mounted. Images were acquired using a Zeiss-LSM710.

Ueda S., Ichiseki T., Shimasaki M., Hirata H., Kawahara N., & Ueda Y. (2021). Glucocorticoid-Induced Osteocytic Cell Death in a Hypoxic Environment Is Associated with Necroptosis. BioChem, 1(2), 98-106.

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Western Blot Analysis of Colon Tissue

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Western blotting analysis was performed as previously described (14) . Fresh colon tissue samples were grounded into powder in liquid nitrogen, and a total of 20 µg of total proteins were fractionated by SDS-PAGE. Proteins were transferred onto PVDF membranes, and membranes were blocked by 5% fat-free milk in Tris-buffered saline containing 0.1% Tween-20 for 1 h at room temperature. Anti-RIP3 (1:1,000; Abcam, Cambridge, UK), anti-MLKL (1:500, Sigma) and anti-β-actin (1:5,000; Sigma) antibodies were diluted in TBS-T and incubated overnight at 4 ℃.
Then, the membranes were washed with horseradish peroxidase-conjugated rabbit anti-mouse IgG, and developed with ECL Plus. β-actin was used as a loading control. Densitometric analysis of the blots was performed using ImageJ software. Average integrated optical density was obtained by analyzing 5 fields of view for each slide evaluated by Image-Pro Plus version 5.0.

Wu T., Dai Y., Xue L., Sheng Y., Xu L, & Xue Y. (2019). Expression of receptor interacting protein 3 and mixed lineage kinase domain-like protein-key proteins in necroptosis is upregulated in ulcerative colitis. Annals of palliative medicine, 8(4).

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Immunohistochemistry of RIP1 and RIP3

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Paraffin-embedded sections were routinely deparaffinized and rehydrated in xylene and different concentrations of ethanol. The sections were then incubated for 10 min with 3% H 2 O 2 to inhibit endogenous catalase. The sections underwent antigen retrieval with sodium citrate buffer for 15 min and were then incubated with anti-RIP1 (1:200, Abcam, Cambridge, Britain) and anti-RIP3 (1:500, Abcam, Cambridge, Britain) antibodies at room temperature for 1 h. The sections were rinsed with PBS and incubated with an appropriate amount of horseradish peroxidase-conjugated secondary antibodies (ZSGB-BIO, Beijing, China) at 37 °C for 30 min. Then, the sections were rinsed with PBS. DAB was used as the chromogen. The slides were detected under 400x magnification by light microscopy with a digital camera and imaging software. The positive cells were colored brown. The number of positive cells was counted in each image. The data were calculated as the percentage of immunohistochemistry-positive cells of the total number of cells per image.

Hao Y., Dong X., Liu H, & Wang Y. (2019). Preconditioning with one-time hydrogen gas does not attenuate skin flap ischemia-reperfusion injury in rat models. Journal of plastic, reconstructive & aesthetic surgery : JPRAS, 72(10).

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Apoptosis and Necroptosis Assessment

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Apoptosis as characterized by DNA fragmentation was measured with a Cell Death Detection ELISA kit (Roche, Basel, Switzerland) [31] . Necrosis/Necroptosis was measured using a lactodehydrogenase (LDH) leakage assay [29, 30] . Moreover, RIP1/3 signaling, a hallmark of necroptosis, was assessed by immunoprecipitation of the RIP1-RIP3 complex [30] . Brie y, cell lysates were immunoprecipitated with anti-human RIP1 (BD Bioscience, Franklin Lakes, NJ) and incubated with protein G agarose beads followed by heat denaturation. The immune complex was then subjected to reducing gel electrophoresis before the proteins were transferred to membranes, which were incubated with anti-RIP1 (1:1000, BD Bioscience) or anti-RIP3 (1:1000, Abcam, Cambridge, UK) for immunoblotting.

Huang C., Pai Y., & Yu L.C. (2021). Divergent Actions of Pyruvate and ATP in Glucose-mediated Irinotecan Chemoresistance in Colorectal Cancer.

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