Tumor cells were lysed on ice with ice-cold radioimmunoprecipitation (RIPA) buffer containing a protease inhibitor cocktail (Sigma-Aldrich) for 30 min. The tumor tissues in RIPA buffer were treated by ultrasound. Then, soluble protein concentrations in lysate were determined by using a BCA protein assay kit (Thermo Scientific, Fremont, CA, USA). Western blot analysis was performed mainly as previously described [21 (link)]. The following antibodies were used: caspase-3, cleaved caspase-3, caspase-8, cleaved caspase-8 (9662S, 9661S, 4790S, 9496S respectively, Cell Signaling Technology, Danvers, MA, USA), RIP1 (610,458, BD Biosciences), pRIP1 (65746S, Cell Signaling Technology, Danvers, MA, USA), RIP3 (13,526, Cell Signaling Technology, Danvers, MA, USA), pRIP3 (ab209384, Abcam, Cambridge, UK), MLKL (ab184718, Abcam, Cambridge, UK), pMLKL (ab187091, Abcam, Cambridge, UK), JNK (9252, Cell Signaling Technology, Danvers, MA, USA), pJNK (4668, Cell Signaling Technology, Danvers, MA, USA), GAPDH (5174S, Cell Signaling Technology, Danvers, MA, USA) and corresponding secondary antibodies (Jackson ImmunoResearch, PA, USA).
P rip3
Manufactured by Abcam
Sourced in United States
P-RIP3 is a recombinant protein that can be used as a positive control or reference standard in various research applications. The core function of this product is to provide a reliable and consistent source of the phosphorylated form of the RIP3 protein for use in experimental procedures.
Automatically generated - may contain errors
Lab products found in correlation
P mlkl, by Abcam (7 mentions)
Caspase 3, by Cell Signaling Technology (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
P rip1, by Cell Signaling Technology (2 mentions)
P rip1, by Abcam (2 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Caspase 8, by Cell Signaling Technology (1 mentions)
P jnk, by Cell Signaling Technology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Ab209384, by Abcam (1 mentions)
Ab187091, by Abcam (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Cleaved caspase 8, by Cell Signaling Technology (1 mentions)
Gapdh, by Abcam (1 mentions)
Hrp conjugated secondary antibodies, by Abcepta (1 mentions)
Ab32503, by Abcam (1 mentions)
Ab56164, by Abcam (1 mentions)
Ab181602, by Abcam (1 mentions)
Ab32124, by Abcam (1 mentions)
Goat anti mouse igg, by Merck Group (1 mentions)
9 protocols using p rip3
1
Tumor Cell Lysis and Western Blot Analysis
2
Evaluation of Necroptosis Pathway Proteins
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Protein was harvested in 1×lysis buffer including a protease inhibitor. 30 µg total protein was separated by 10% SDS-PAGE, transferred to PVDF membrane, blocked for 1 h with TBST containing 5% BSA, and incubated at 4 °C overnight with the appropriate primary antibodies: RIP3 (Abcam, ab56164, 1:1,000), pRIP3 (Abcam, ab222320, 1:1,000), MLKL (CST, #37705, 1:1,00), p-MLKL (CST, #37333, 1:10,00), Bax (Abcam, ab32503, 1:1,000), Bcl-2 (Abcam, ab32124, 1:1,000), and GAPDH (Abcam, ab181602, 1:20,000). They were then incubated with HRP-conjugated secondary antibodies (Abgent, 1:30,000), which was followed by chemiluminescent substrate development (Bio-Rad).
3
Immunofluorescence of Cell Death Markers
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Liver tissues were cut, deparaffinized, dehydrated, and incubated with specific antibodies against p-RIP3 (Abcam, USA), p-MLKL (Abcam, USA) and CD68 (Abcam, USA) overnight at 4 °C. Subsequently, the sections were washed thrice with 1 × PBS and then incubated with fluorochrome-conjugated secondary antibody for 60 min at room temperature in the dark. 4, 6-Diamidino-2-phenylindole was finally applied to dye the nucleus. A fluorescence microscope DM5000 B (Leika, Germany) was used for analysis.
4
Quantitative Western Blot Analysis
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Western blotting was performed according to a standard method, as described previously [22 (link)]. The following primary antibodies were used in this study: RIP1 (1 : 2000, cell signaling, 45726, USA), p-RIP1 (1 : 2000, cell signaling, 53286, USA), RIP3 (1 : 2000, Abcam, ab226297, USA), p-RIP3 (1 : 2000, Abcam, ab195117, USA), MLKL (1 : 2000, Abcam, ab243142, USA), p-MLKL (1 : 2000, Abcam, ab187091, USA), Caspase3 (1 : 2000, cell signaling, 9662, USA), cleaved Caspase3 (1 : 2000, cell signaling, 9664, USA), DOR (1 : 2000, Millipore, AB1560, USA), and β-actin (1 : 2000, Sigma, A5441, USA). Goat anti-rabbit IgG (1 : 4000) and goat anti-mouse IgG (1 : 4000) were purchased from Sigma. The protein bands were quantified by densitometry using ImageJ software, and protein expression was normalized to the expression of the internal control.
5
Protein Expression and Localization in Brain Tissue
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Total proteins were harvested from the cortex and hippocampus. The proteins were transferred to polyvinylidene fluoride membranes (Millipore) that were blocked with 5% bovine serum albumin (BSA) for 1 hour at room temperature and incubated overnight with the following primary antibodies at 4℃: anti‐p‐RIP3 (Abcam), anti‐p‐MLKL (Abcam), anti‐tumour necrosis factor‐α (TNF‐α) (Abcam), anti‐IL‐1β (Abcam), anti‐IL‐6 (Abcam) and anti‐β‐actin (Cell Signaling Technology). After incubation with secondary antibodies (Solarbio), the positive bands were visualized using an ECL substrate (Solarbio). For immunohistochemical analysis, brain tissue sections were incubated overnight with the following primary antibodies at 4℃: anti‐p‐RIP3 (Abcam) and anti‐p‐MLKL (Abcam). Subsequently, a PV9000 kit (ZSGB‐BIO) was used for follow‐up. For immunofluorescence analysis, brain tissues were blocked with goat serum for 1 hour and incubated with primary antibodies against p‐RIP3 (Abcam), p‐MLKL (Abcam), NeuN (Abcam), Iba‐1 (Wako; Servicebio) and GFAP (Servicebio) overnight at 4℃. Then, the tissues were incubated with Alexa Fluor® 594 goat anti‐rabbit IgG and Alexa Fluor® 488 goat anti‐mouse IgG secondary antibodies (Thermo Fisher Scientific). Nuclei were counterstained with DAPI (Thermo Fisher Scientific).
6
Cellular Death Pathway Assays
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Tryptic soya broth (TSB) and agar (TSA) were procured from Hi-Media Laboratories. Sodium dodecyl sulfate (SDS), lysozyme, necrostatin-1, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), metformin hydrochloride, 2-Deoxy-d -glucose, dihydrorhodamine 123, cytochalasin D, Durcupan™ ACM resin components; A, B, C, and D, triton X-100, absolute ethanol, and Bradford’s reagent were purchased from Sigma-Aldrich. 1-octanol, tris-buffer, chloroform, HCl, and luminata forte western HRP substrate were obtained from Merck-Millipore. Fetal bovine serum (FBS), TRIzol Reagent, and penicillin–streptomycin solution, and were purchased from Gibco-Invitrogen. CellTiter-Glo luminescent cell viability assay kit for ATP detection and CytoTox-ONE™ homogeneous membrane integrity assay kit for estimating LDH release were obtained from Promega and the manufacturer’s instructions were followed. DyNAmo ColorFlash SYBR Green qPCR kit and Verso cDNA synthesis kit were procured from Thermo Fisher Scientific. RIP1, RIP3, and β-actin antibodies were purchased from Cell Signaling Technologies, MLKL, pMLKL, and pRIP3 were procured from Abcam. Secondary HRP antibodies were obtained from Santa Cruz Biotechnology. All plastic wares for cell culture were obtained from Nunc. Millipore water (Type II) was used in all the experiments.
7
Molecular Mechanisms of Necroptosis
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Shikonin, Cyclosporine A (CsA), Nec-1, GSK872 and MnTBAP were purchased from selleckchem company (Houston, TX). Shikonin was dissolved in PBS to a storage concentration of 50 mmol/L. The primary antibodies against the following proteins RIP1, p-RIP1, RIP3, p-RIP3, Cyclophilin A, AIF, DKAPKcs, p-DNAPKcs, ATM, p-ATM, p-H2AX, H2A, NRF2, p-NRF2 antibodies were all purchased from Abcam company (Cambridge, MA). Anti-β-Actin antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). Other reagents were purchased from Sigma (St. Louis, MO).
8
Necroptosis Pathway Inhibitors Evaluation
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The following materials have been used: human recombinant soluble TNFα (Genentech, South San Francisco, CA, USA), mouse recombinant soluble TNFα (R&D, Minneapolis, MN, USA), Flag-TNFα (Enzo, Life Sciences, Farmingdale, NY, USA), zVAD-Fmk (MBL, Woburn, MA, USA), Nec-1 (Sigma, St. Louis, MO, USA), BV6, Nec-1a, Nec-4, Nec-1Cl, GSK84336 (link), 37 (link), 38 (link) (all synthesized at Genentech). The primary antibodies used were directed against: RIP1, FADD, JNK (Jun N-terminal kinase), P-JNK, P-ERK (BD Biosciences), human and mouse RIP3 (Imgenex, Littleton, CO, USA, Genentech and Cell Signaling, Danvers, MA, USA), human c-IAP1 and mouse pan-c-IAP (R&D), human c-IAP2 (Novus, Littleton, CO, USA), actin (Sigma), mouse FADD (Santa Cruz Biotechnology, Santa Cruz, CA, USA and Genentech), ubiquitin (P4D1; Cell Signaling), anti-human P-RIP1 antibody raised against S166 of human RIP1 (Cell Signaling), IκBα, P-IκBα, P-p38, Hsp90 (Cell Signaling), human caspase-8 (Cell signaling and Enzo), mouse caspase-8, mouse c-IAP1 (Enzo), Myc, K63 and linear ubiquitin chains (Genentech), MLKL (Millipore, Billerica, MA, USA), P-RIP3 and P-MLKL (Abcam, Cambridge, MA, USA), FLAG-HRP conjugate (Sigma). Plasmid transfections were carried out using Fugene 6 (Promega) for 24 or 48 h.
9
Western Blot Analysis of Cell Signaling
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Western blotting was performed as previously described.50 (link) Briefly, the harvested cells were lysed using a Minute Total Protein Extraction Kit (Invent Biotechnologies, #SD001) containing 1% protease inhibitor cocktail (Sigma-Aldrich, #P8340). The protein samples were boiled for 10 min at 70 °C with loading buffer, electrophoresed on SDS-PAGE (Bio-Rad, #4561094) and then transferred to a PVDF membrane. Subsequently, the membranes were blocked with 10% (w/v) nonfat milk in TBST (TBS containing 0.1% Tween-20) for 1 h at room temperature. The blots were incubated overnight at 4°C with primary antibodies. After the membranes were washed with TBST, they were further incubated with secondary antibodies for 1 h at room temperature. The blots were visualized using an ECL kit according to the manufacturer’s instructions. The intensity of the bands was semiquantified using ImageJ software. The following antibodies were used for Western blotting: p-RIP3 (CST, #93654), RIP3 (CST, #13526), p-RIP1 (CST, #65746), RIP1 (CST, #3493), p-MLKL (CST, #91689), MLKL (CST, #14993), vinculin (CST, #13901), horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (CST, #7076S), HRP-conjugated anti-mouse IgG (CST, #7072),and Metallothionein (Abcam, #ab12228).
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