Phospho rip3

Manufactured by Cell Signaling Technology

Sourced in United States

Phospho-RIP3 is a lab equipment product that detects phosphorylated receptor-interacting serine/threonine-protein kinase 3 (RIP3). RIP3 is a key regulator of necroptosis, a form of programmed cell death. The Phospho-RIP3 product is designed to enable the study of RIP3 phosphorylation, which is a critical event in the necroptosis signaling pathway.

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Phospho-RIP3 (Thr231/Ser232) (2 citations)

5 protocols using phospho rip3

Extraction and Isolation of Oleanolic Acid

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Cisplatin, sodium thiosulfate (ST), oleanolic acid (OA), necrostatin-1 (Nec-1), and z-VAD-FMK were purchased from Sigma-Aldrich (St. Louis, MO, USA). OAA was purified from V. angularis as previously described [36 (link)]. Briefly, the V. angularis material was dried, pulverized to a fine powder, and extracted twice with 95% ethanol (EtOH) at 70 °C. The EtOH extracts were concentrated under reduced pressure. For fractionation, the EtOH extract was resuspended in water and then extracted with ethyl acetate (EtOAc). To isolate compound from the EtOAc fraction, it was further chromatographed on silica gel using a gradient hexane-EtOAc solvent system. The recrystallization of H3 in EtOAc yielded OAA. The primary antibodies RIP (#3493, rabbit monoclonal), phospho-RIP (#65746, rabbit monoclonal), RIP3 (#95702, rabbit monoclonal), phospho-RIP3 (#91702, rabbit monoclonal), MLKL (#37705, rabbit monoclonal), phospho-MLKL (#37333, rabbit monoclonal), and β-actin (#4967S, rabbit monoclonal), as well as the anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody (#7074S) and anti-mouse IgG horseradish peroxidase-conjugated secondary antibody (#7076S), were purchased from Cell Signaling Technology (Danvers, MA, USA).

Lee B., Kim Y.Y., Jeong S., Lee S.W., Lee S.J., Rho M.C., Kim S.H, & Lee S. (2024). Oleanolic Acid Acetate Alleviates Cisplatin-Induced Nephrotoxicity via Inhibition of Apoptosis and Necroptosis In Vitro and In Vivo. Toxics, 12(4), 301.

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Immunoblotting of Apoptosis-Related Proteins

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Protein lysates were extracted using ice‐cold lysis buffer (1% NP‐40, 1mM DTT, supplemented with protease and phosphatase inhibitors in PBS) and subjected to immunoblotting. Primary antibodies against APP and β‐actin were obtained from Biolegend, and Santa Cruz Biotechnology Inc, respectively. RIP, phospho‐RIP, MLKL, phospho‐MLKL, RIP3 and phospho‐RIP3 were purchased from Cell Signalling Technology Inc. The dilution ratio used for each antibody was shown in Table S5. All images were captured using the ChemiDocTM XRS+molecular imager (Bio‐Rad Laboratories).

Chan H., Leong C., Lim C, & Koh R. (2022). Roles of receptor‐interacting protein kinase 1 in SH‐SY5Y cells with beta amyloid‐induced neurotoxicity. Journal of Cellular and Molecular Medicine, 26(5), 1434-1444.

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Immunofluorescent Staining of Macrophages

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Macrophages were fixed for 2 h with 4% paraformaldehyde in PBS at 24 h post‐infection. Cells were then washed three times with PBS and permeabilized for 15 min with 2% BSA, 2% saponine, 0.1% Triton x‐100 in PBS at RT. After three washes with PBS, samples were blocked for 1 h with 2% BSA in PBS at RT. Rabbit primary antibodies anti‐cleaved Caspase‐8 (Cell Signaling), ASC/TMS1 (Cell Signaling), cleaved Caspase‐1 (Cell Signaling), phospho‐RIP3 (Cell Signaling), phospho‐MLKL (Cell Signaling), mouse CD45.1‐AlexaFluor647 (BioLegend) were diluted 1:300 in 2% BSA in PBS and used for primary staining of the cells overnight at 4°C. After three washes in 2% BSA in PBS, samples (excluding those stained with anti‐mouse CD45.1‐AlexaFluor647) were incubated with a secondary goat anti‐rabbit antibody conjugated to Alexa Fluor 647 (ThermoFisher Scientific) diluted 1:300 in 2% BSA in PBS for 1 h at RT. Samples were stained with 1:1,000 Hoechst (ThermoFisher Scientific) and stored in PBS at 4°C before imaging by fluorescence microscopy.

Toniolo C., Dhar N, & McKinney J.D. (2023). Uptake‐independent killing of macrophages by extracellular Mycobacterium tuberculosis aggregates. The EMBO Journal, 42(9), e113490.

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Western Blot Analysis of Necroptosis Markers

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Western bolt experiments were performed with a primary antibody for TLR4 (Cell Signaling, 2219), Cleaved caspase-8 (Cell Signaling, 8592), RIP3 (ProScience, 2283), phospho RIP3 (Cell Signaling, 57220), MLKL (Santa Cruz Biotech, sc-165025), phospho MLKL (Cell Signaling, 37333), TNFR1 (Santa Cruz Biotech, sc8436) and βactin (cell signaling, 4970/58169) overnight at 4 °C coupled with appropriate secondary antibody as previously described^{3 (link)}. ECL was used as a method of detection. Chemiluminescence was recorded with an Image Station 440CF and results analyzed with the 1D Image Software (Kodak Digital Science, Rochester, NY).

Jain S., Plenter R., Nydam T., Gill R.G, & Jani A. (2021). Deletion of TLR4 reduces apoptosis and improves histology in a murine kidney transplant model. Scientific Reports, 11, 16182.

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Mechanistic Study of Necroptosis Pathway

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Dexamethasone (#D4902) and 2,4-dinitrochlorobenzene (DNCB, #138630) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Castor oil and acetone were purchased from Aladdin Chemical (Shanghai, China) and Sinopharm Chemical Reagent (Shanghai, China), respectively. Propidium iodide (PI) / RNase staining solution (#4087 S) was purchased from Cell Signaling Technology (Danvers, MA, USA). In this study, some antibodies were purchased from Cell Signaling Technology, including phospho-RIP (#65746 for human and #38662 for mouse), RIP3 (#95702), phospho-RIP3(#91702), MLKL (#37705), phospho-MLKL (#37333), GAPDH (#5174), and goat anti-rabbit IgG HRP-linked antibody (#7074). Other antibodies were purchased from Abcam (Cambridge, MA, USA), including RIP (#ab125072, #ab72139), RIP3 (#ab226297), phospho-RIP3 (#ab209384), MLKL (#ab184718), phospho-MLKL (#ab187091), CD4 (#ab183685), CD8 (#ab217344), MPO (#ab208670), MMP-9 (#ab283575) and NE (#ab68672). Tumor necrosis factor α (TNF-α), Smac-mimetic compound, z-VAD-fmk and necrostatin-1 (as described previously [27 (link)]) were gifts from Dr. Sudan He’s Lab in Suzhou Institute of Systems Medicine. TNF-α and IFN-γ used to simulate the ACD microenvironment in HaCaT were purchased from R&D system (Minneapolis, USA) and PeproTech (New Jersey, USA). rHMGB1 was purchased from R&D system (Minneapolis, USA).

Lian N., Chen Y., Chen S., Xiao T., Song C., Ke Y., Wei X., Gong C., Yu H., Gu H., Chen Q., Li M, & Chen X. (2022). Necroptosis-mediated HMGB1 secretion of keratinocytes as a key step for inflammation development in contact hypersensitivity. Cell Death Discovery, 8, 451.

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