Whole cell extracts (WCEs) were prepared in RIPA buffer. Protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktail (Sigma) were included in lysis buffer. Secreted proteins in culture media were precipitated by trichloroacetic acid. For immunoprecipitation, anti-RIPK3 (Prosci) or anti-Caspase 8 (Enzo Life Sciences) antibodies were used. Western blotting was performed with anti-RIPK1 (BD Biosciences), RIPK3 (Prosci), FADD (kindly provided by A. Winoto), Caspase 8 (Enzo Life Sciences), MyD88 (eBioscienes), IL-1β, cIAP1/2 (R&D systems), GFP (Roche), Caspase-1 (Santa Cruz), and IL-18 (BioVision) antibodies. Anti-HSP90 antibody (BD Biosciences) was used as a loading control.
Anti ripk3
Manufactured by ProSci
Sourced in United States
Anti-RIPK3 is a laboratory reagent that binds to and inhibits the RIPK3 protein. RIPK3 is a key regulator of necroptosis, a form of programmed cell death. This product can be used in research applications to study the role of RIPK3 and necroptosis in various biological processes.
Automatically generated - may contain errors
Lab products found in correlation
Anti ripk1, by BD (7 mentions)
Anti ripk1, by Cell Signaling Technology (3 mentions)
Anti cd9, by System Biosciences (3 mentions)
Anti cd63, by System Biosciences (3 mentions)
Anti cd81, by System Biosciences (3 mentions)
Anti p mlkl, by Abcam (2 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 ripk3, by Santa Cruz Biotechnology (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)
Phosphatase inhibitor cocktail, by Merck Group (1 mentions)
9 protocols using anti ripk3
1
RIPK3 and Caspase-8 Immunoprecipitation
2
Immunoblotting Reagents for Cell Death Signaling
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Antibodies and other reagents were obtained as follows: anti-AF6 and anti-RIPK1(receptor-interacting serine/threonine kinase 1) antibodies from BD Transduction Laboratories; anti-pRIPK1(S166), anti-TNFR1(TNF receptor superfamily member 1A), anti-Hsp90(heat shock protein 90), and anti-GAPDH(glyceraldehyde-3-phosphate dehydrogenase) antibodies from Cell Signaling Technology; anti-pRIPK3(Ser232) (receptor-interacting serine/threonine kinase 3), anti-pMLKL(Ser345)(mixed lineage kinase domain-like pseudokinase), anti-MLKL and anti-CYLD(CYLD lysine 63 deubiquitinase) antibodies from abcam; anti-USP21, anti-FADD (Fas-associated via death domain), anti-TRAF2(TNF receptor-associated factor 2), and anti-TRADD(TNFRSF1A associated via death domain) antibodies from Santa Cruz Biotechnology; anti-A20(TNF alpha-induced protein 3) antibody from ABClonal; anti-RIPK3 from Prosci; and anti-actin antibody from Servicebio. Smac was a gift from Haibing Zhang’s lab. Other reagents were purchased as follows: mouse TNFα (Minneapolis), lipopolysaccharide (LPS) (Sigma-Aldrich Cat. L4516), Nec-1 (Enzo Life Science), Z-Val-Ala-Asp (OMe)-FMK (z-VAD) and Bv6 (MedChemExpress).
3
RIPK1 and RIPK3 Protein Complexes
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Brain homogenates from a block of tissue containing the entire cerebral contusion were lysed in RIPA buffer containing protease and phosphatase inhibitors (Sigma-Aldrich), followed by sonication and centrifugation at 15,000 × g for 30 min. Supernatants containing protein complexes were incubated with anti-RIPK3 (Prosci, #2283) or anti-RIPK1 (BD bioscience, #610458) antibodies conjugated to Magnetic Protein G Dynabeads (Thermo Fisher Scientific) at 4 °C overnight. Beads were washed three times with washing buffer and proteins were eluted in 1X loading buffer and processed/ for western blot.
4
RIPK1-Mediated Necroptosis Signaling
5
RIPK1-Mediated Necroptosis Signaling
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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 described19 (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).
Signaling studies in MEFs were performed as described19 (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).
6
Apoptosis-Inducing Agents and Antagonists
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Drugs used in this study were recombinant murine sTRAIL/Apo2L (Peprotech; NJ, USA), murine TNFα (Peprotech), Debio1143/AT-406 (Selleck Chemicals; Texas, USA), LCL161 (Selleck Chemicals), SM-164 (ApexBio; Texas, USA), Birinapant (ApexBio), BV6 (ApexBio), GDC-0512 (Selleck), doxorubicin (Sigma-Aldrich; MO, USA) and cisplatin (Sigma-Aldrich). The following antibodies were used: anti-cIAP1 (Enzo Life sciences; NY, USA), anti-cIAP2 (R&D systems; MN, USA), anti-XIAP (MBL International; MA, USA), anti-FADD (Abcam; Cambridge, UK), anti-TNFR1 (Abcam), anti-TRAIL-R2 (R&D systems), anti-caspase-3 (BD Biosciences; NJ, USA), anti-caspase-8 (Abcam), anti-RIPK1 (BD Biosciences), anti-RIPK3 (ProSci; CA, USA), anti-MLKL1 (Abcam), anti-phosphoS345-MLKL (Abcam), anti-CYLD (Thermo Fisher Scientific; Massachusetts, USA), anti-crmA (Santa Cruz Biotechnology; Texas, USA), rabbit anti-GFP (polyclonal in-house antibody), goat anti-rabbit FITC (Merck Millipore; MA, USA), rabbit anti-goat-FITC (Thermo Fisher Scientific), mouse anti-GAPDH (Merck Millipore), donkey anti-rabbit HRP conjugated antibody (GE Healthcare Life Sciences; NJ, USA), goat anti-rat HRP conjugated antibody (GE Healthcare Life Sciences), and rabbit anti-mouse-HRP conjugated antibody (Sigma).
7
Western Blot Analysis of Cell Death Proteins
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Samples were loaded onto a 10% SDS‐PAGE gel and then transferred to a polyvinylidene fluoride membrane. Binding of the primary antibody was detected by peroxidase‐conjugated secondary antibodies and enhanced chemiluminescence. The sources of antibodies and dilutions used for Western blot analysis were as follows: anti‐RIPK3(1:1000, ProSci, Cat #:2283), anti‐RIPK3(1:1000, Cell Signaling Technology, Cat#:13526), anti‐MLKL (1:1000, EMD Millipore, Cat#MABC604), anti‐RIPK1(1:500, BD Biosciences, Cat#:610458), anti‐CD63(1:1000, System Biosciences, Cat#:EXOAB‐CD63A‐1), anti‐TSG101(1:1000, Abcam, Cat#:ab30871), anti‐CD81(1:1000, System Biosciences, Cat#:EXOAB‐CD81A‐1), anti‐CD9(1:1000, System Biosciences, EXOAB‐CD9A‐1), anti‐Calnexin (1:1000, Cell Signalling Technology, Cat#:2433S).
8
Western Blot Analysis of RIPK1, RIPK3, and p-MLKL
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Lysates of heart tissue and cultured cells were prepared in RIPA buffer (Solarbio) supplemented with a protease inhibitor cocktail (Abmole, Houston, USA). The total protein content (80 μg) was segregated on 12% SDS–polyacrylamide gels and was then transferred onto 0.45 μm nitrocellulose membranes (Millipore, Plano, TX, USA). After being blocked in PBS-T buffer containing 5% fat-free milk at 37 °C for 2 h, the membranes were incubated with rabbit anti-RIPK1 (1:1200, Cell Signaling, Danvers, USA), anti-RIPK3 (1:2000, ProSci, San Diego, USA), anti-p-MLKL (1:2000, Abcam, Cambridge, MA, USA) or anti-GAPDH (1:2000, Abcam) antibodies at 4 °C overnight. After incubation with the horseradish peroxidase-conjugated secondary antibodies, the protein bands were visualized using an ECL kit (Sigma, USA).
9
Inhibition of Akt and Necroptosis Pathways
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The following materials were purchased from commercial companies: TNFα (PeroTech; Rocky Hill, NJ, USA); pan-caspase inhibitor z-VAD-fmk (Abcam, Cambridge, MA, USA). InSolution Akt Inhibitor viii isozyme-selective, Akti-1/2 and InSolution rapamycin were obtained from Calbiochem (San Diego, CA, USA). MitoSox Red was obtained from Invitrogen (Carlsbad, CA, USA). Hoechst 33258, butylated hydroxyanisole (BHA) and rotenone were obtained from Sigma (St. Louis, MO, USA). Nec-1 (5-(7-chloro-1H-indol-3-ylmethyl)-3-methylimidazolidine-2,4-dione), the inactive analog of necrostatin-1 analog (Nec-1i; 5-(7-chloro-1H-indol-3-ylmethyl)imidazolidine-2,4-dione),3 (link), 12 (link), 13 (link) was a kind gift from Dr. Greg Cuny. LOX-1 was obtained from Chembridge (compound 5680672; San Diego, CA, USA), and Bai was from Cayman Chemicals (Ann Arbor, MI, USA). Antibodies were obtained from commercial sources: anti-pAkt-473, anti-p–Akt-308, anti-p-GSK-3β, anti-p-mTOR, anti-p-S6, anti-mTOR, anti-Akt1, anti-Akt2, anti-Akt3, and anti-Akt from Cell Signaling (Danvers, MA, USA); anti-RIPK1 from BD Transduction Laboratories (Lexington, KY, USA); anti-RIPK3 from ProSci Incorporated (San Diego, CA, USA); anti-HMGB1 and β-actin were obtained from Abcam.
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