Anti ripk3

Manufactured by Santa Cruz Biotechnology

Sourced in United States

Anti-RIPK3 is a laboratory-grade antibody that specifically targets the RIPK3 protein. RIPK3 is a key regulator of necroptosis, a form of programmed cell death. The antibody can be used for the detection and study of RIPK3 in various experimental systems.

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Lab products found in correlation

Anti ripk1, by Cell Signaling Technology (3 mentions) Nec 1, by Merck Group (2 mentions) Anti bim, by BD (2 mentions) Anti bclx, by BD (2 mentions) Anti bcl 2, by Merck Group (2 mentions) Anti a20, by Cell Signaling Technology (2 mentions) Anti k63, by Merck Group (2 mentions) Dynabead protein a, by Thermo Fisher Scientific (2 mentions) Anti ubiquitin, by Santa Cruz Biotechnology (2 mentions) Anti bax, by Santa Cruz Biotechnology (2 mentions) Dynabeads m 270 epoxy, by Thermo Fisher Scientific (2 mentions) Celltiter glo luminescent cell viability assay, by Promega (2 mentions) β actin, by Merck Group (2 mentions) Anti ripk1, by BD (2 mentions) Anti ripk3, by ProSci (2 mentions) Lsm 700, by Zeiss (2 mentions) Protein a g agarose bead, by Santa Cruz Biotechnology (1 mentions) Dylight 594, by EarthOx (1 mentions) Anti ripk3 antibody, by Santa Cruz Biotechnology (1 mentions) Anti rl2, by Santa Cruz Biotechnology (1 mentions)

Close spelling variants of this product

RIPK3 (11 citations) Anti-RIPK3 antibody (2 citations)

6 protocols using anti ripk3

RIPK1-Mediated Necroptosis Signaling

Cited 1 time

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Stimulated T cells were incubated in lysis buffer. For signaling complex analyses, cell lysates were pre-cleared with Protein G beads prior to immunoprecipitation with anti-RIPK1 antibody. Antibodies and reagents used for immunoprecipitation and immunoblotting studies included: anti-RIPK1, anti-Bim, anti-Bclx, (BD Biosciences), nec1, anti-Bcl2, β-actin (Merck chemicals), anti-RIPK3 (ProSci Incorporated), anti-ubiquitin, anti-Bax, anti-RIPK3 (Santa Cruz Biotechnology), anti-RIPK1, anti-A20 (Cell signaling Technology), QVD, ZVAD (Enzo Life Science).
Signaling studies in MEFs were performed as described^{19 (link)}. RIPK1 immunoprecipitations were performed using anti-RIPK1 (Cell Signaling) and Dynabeads M270 Epoxy (Invitrogen). K63-ubiquitin immunoprecipitations were performed using anti-K63 (Millipore) and Dynabead Protein A (Invitrogen). Studies of RIPK3 mutants in A20^−/−Ripk3^−/− MEFs were performed by generating RIPK3 lysine mutants, introducing mutant RIPK3-encoding cDNAs into GFP expressing lentiviral constructs, and infecting A20^−/−Ripk3^−/− MEFs with these viruses. Productively infected cells were FACS-sorted to obtain pure populations of RIPK3-expressing cells prior to being tested in necroptosis assays. Cell survival of MEFs was quantitated using the CellTiter-Glo Luminescent Cell Viability Assay per manufacturer’s instructions (Promega).

Onizawa M., Oshima S., Schulze-Topphoff U., Oses-Prieto J.A., Lu T., Tavares R., Prodhomme T., Duong B., Whang M.I., Advincula R., Agelidis A., Barrera J., Wu H., Burlingame A., Malynn B.A., Zamvil S.S, & Ma A. (2015). The ubiquitin-modifying enzyme A20 restricts the ubiquitination of RIPK3 and protects cells from necroptosis. Nature immunology, 16(6), 618-627.

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Immunoprecipitation of RIPK3 in Mouse Skin

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Tissues of mouse back skin samples were lysed with pre-chilled IP buffer, and the supernatant was collected by centrifugation as described previously^{28 (link)}. Part of the lysate was saved as input. Protein A/G agarose beads (Santa Cruz, CA, USA) were added to the remaining lysis content and incubated for 1 h at 4 °C. Supernatant was collected by centrifugation. 1.0 μg anti-RIPK3 (1:300; Santa Cruz, CA, USA) was added and incubated at 4 °C overnight, and normal mouse IgG as a negative control. Protein A/G agarose beads were again added and incubated for 4 h. The immunoprecipitated complexes were collected by centrifugation and washed four times with 1 ml pre-chilled IP buffer, followed by 40 μl loading buffer. Western blot analysis was performed on the samples as previously described.

Duan X., Liu X., Liu N., Huang Y., Jin Z., Zhang S., Ming Z, & Chen H. (2020). Inhibition of keratinocyte necroptosis mediated by RIPK1/RIPK3/MLKL provides a protective effect against psoriatic inflammation. Cell Death & Disease, 11(2), 134.

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Immunofluorescence Staining of RIPK1 and RIPK3

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The cells were fixed with 4% paraformaldehyde for 10 min at room temperature and then permeabilized with 0.2% Triton X-100 for 10 min at room temperature. The cells were blocked with 3% BSA (Sigma-Aldrich, St Louis, MO, USA) at room temperature for 30 minutes. Next, the cells were incubated with anti-RIPK1 (Cell Signaling Technology, Inc., USA) and anti-RIPK3 (Santa Cruz Biotechnology, CA) antibodies for 2 hours at 37 °C. Then, the cells were rinsed with PBS and incubated with Dylight 594 and DyLight 488 AffiniPure secondary antibodies (EarthOx, San Francisco, CA, USA) for 1 hour. Next, the cells were counterstained with DAPI for 15 minutes at 37 °C in the dark. After being washed with PBS, the cells were visualised by confocal microscopy (Carl Zeiss LSM700).

Tian F., Yao J., Yan M., Sun X., Wang W., Gao W., Tian Z., Guo S., Dong Z., Li B., Gao T., Shan P., Liu B., Wang H., Cheng J., Gao Q., Zhang Z., Cao W, & Tian Y. (2016). 5-Aminolevulinic Acid-Mediated Sonodynamic Therapy Inhibits RIPK1/RIPK3-Dependent Necroptosis in THP-1-Derived Foam Cells. Scientific Reports, 6, 21992.

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Co-immunoprecipitation Protocol for Protein-Protein Interactions

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Co-immunoprecipitation (Co-IP) analysis was as described previously [51 (link)]. The heart tissues were homogenate in modified RIPA buffer by sonication and frozen on dry ice for 60 min. Residual myocardial debris was removed by centrifugation at 18,000 × g, 4° C for 15 min. Protein concentration was measured, 50 μg of total protein was used for immunoprecipitation, 25 μg was used as an input control. Then, each IP reaction was adjusted to a total volume of 300 μl in microfuge tubes by the addition of lysis buffer containing protease and phosphatase inhibitors. 2 μl of anti-O-GlcNAc antibody (RL2) or anti-RIPK3 antibody (Santa Cruz Co., California, USA) was added. Tubes were incubated for approximately 15 h at 4° C on a rotor. Subsequently, 50 μl of a 50:50 slurry of protein A/G plus-agarose beads (Santa Cruz Co., California, USA) was added, followed by incubation at 4° C overnight. The beads were resuspended in LDS buffer that contained 50 mmol/L DTT. Finally, the resulting co-IP reaction and input controls were detected by western blotting with anti-RIPK3 (1:1000 dilution, Santa Cruz Co., California, USA), anti-RL2 (1:2000 dilution, Santa Cruz Co., California, USA) and anti-MLKL (1:1000 dilution, Santa Cruz Co., California, USA).

Zhang J., Yu P., Hua F., Hu Y., Xiao F., Liu Q., Huang D., Deng F., Wei G., Deng W., Ma J., Zhu W., Zhang J, & Yu S. (2020). Sevoflurane postconditioning reduces myocardial ischemia reperfusion injury-induced necroptosis by up-regulation of OGT-mediated O-GlcNAcylated RIPK3. Aging (Albany NY), 12(24), 25452-25468.

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Comprehensive Immunohistochemical Analysis of Retina and Brain

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Cross-sections of the retina or brain and whole-mount retinas for immunohistochemical analysis were firstly washed several times with PBS, and then blocked in PBS containing 5% goat serum and 1% Triton x-100 for 1h at room temperature. The blocked tissues were incubated with the following primary antibodies overnight at 4°C: anti-β-amyloid, clone 6E10 (1:500; BioLegend), anti-β-amyloid, clone 4G8 (1:500; BioLegend), anti-rhodopsin, clone 1D4 (1:1000; Santa Cruz), anti-M-opsin (1:500; Millipore), anti-S-opsin (1:500; Millipore), anti-PKCα (1:200; Abcam), anti-PKCα (1:100; Invitrogen), anti-CtBP2 (1:200; BD), anti-PSD95 (1:100; CST), anti-ZO-1 (1:100; Invitrogen), anti-RPE65 (1:200; Novus), anti-p16^ink4α (1:100; Proteintech), anti-p21 (1:100; Santa Cruz), anti-cleaved caspase3 (1:100; CST), anti-RIPK3 (1:100; Santa Cruz), anti-pRIPK3 (1:400; CST) and anti-pMLKL (1:100; Abcam). AlexFluro-488 or -555 conjugated secondary antibody (1:500; CST) incubation were performed for 2.5 h at room temperature. Thereafter, DAPI (1:1000, Invitrogen) were used to label the nuclear for 10 min at room temperature.

Zhang J., Gao F., Ma Y., Xue T, & Shen Y. (2021). Identification of early-onset photoreceptor degeneration in transgenic mouse models of Alzheimer's disease. iScience, 24(11), 103327.

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RIPK1-Mediated Necroptosis Signaling

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