AML12 cells were treated with 0.3% Triton (Triton X-100, Dilution with PBS) for 15 min. 10% Normal goat serum was dripped on to the slide, which was then sealed at room temperature for 30 min. The slides were then incubated overnight in the primary antibodies at 4°C. PBST was used to wash three times before incubation with second antibody for 30 min at room temperature. The nuclei were stained with DAPI and the following antibodies were used to highlight other cellular components: anti-RIP3 (Santa-Cruz, sc-374639), goat antimouse IgG H&L (Alexa Fluor® 488) (Abcam, ab150117). 12 mice were examined in each group, and 3 independent view fields per section were quantified.
Anti rip3
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Anti-RIP3 is a laboratory product that specifically binds to the RIP3 (receptor-interacting serine/threonine-protein kinase 3) protein. RIP3 is a key regulator of necroptosis, a form of programmed cell death. Anti-RIP3 can be used in research applications to study the role of RIP3 and necroptosis in various biological processes.
Automatically generated - may contain errors
Lab products found in correlation
Anti rip1, by Santa Cruz Biotechnology (4 mentions)
Anti bax, by Santa Cruz Biotechnology (2 mentions)
Anti akt, by Cell Signaling Technology (2 mentions)
Anti β actin, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Ecl kit, by Thermo Fisher Scientific (2 mentions)
Goat anti mouse igg h l alexa fluor 488, by Abcam (1 mentions)
Anti bcl 2, by Santa Cruz Biotechnology (1 mentions)
Mayer s hematoxylin, by Merck Group (1 mentions)
Tp 125 hl, by Thermo Fisher Scientific (1 mentions)
Dimethylaminopyridine, by Merck Group (1 mentions)
P selectin, by Santa Cruz Biotechnology (1 mentions)
Phospho pi3 kinase p85, by Cell Signaling Technology (1 mentions)
Anti myeloperoxidase, by Abcam (1 mentions)
1 3 dimethylaminopropyl 3 ethylcarbodiimide hydrochloride, by Merck Group (1 mentions)
Celastrol, by Meilun (1 mentions)
Cleaved caspase 3 asp175, by Cell Signaling Technology (1 mentions)
Pi3 kinase p85α, by Cell Signaling Technology (1 mentions)
Phospho mtor, by Cell Signaling Technology (1 mentions)
Phospho akt, by Cell Signaling Technology (1 mentions)
12 protocols using anti rip3
1
Immunostaining of RIP3 in AML12 cells
2
Immunocytochemistry Analysis of Ru and Ir Complexes
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After
determining the appropriate dose, Vero and HepG2 cells were seeded
for immunocytochemistry analysis, and theRu1, Ru2 , and Ir1 complexes were applied to the cells. The cells were then
fixed with 4% paraformaldehyde (Merck, 30525) and washed thrice with
phosphate-buffered saline (PBS, Bioshop, PBS404.100). Later, the cells
were treated with Triton X-100 (AppliChem, A4975) on ice for permeabilization
and washed with PBS. Afterward, endogenous peroxidase activity was
quenched by incubation with 3% H2O2 (Merck,
107209) for 5 min at room temperature. The cells were then washed
with PBS and incubated overnight at 4 °C with the following primary
antibodies: anti-Bax (Santa Cruz, sc-526), anti-Bcl2 (Santa Cruz,
sc-7382), anticaspase 3 (Novus Biologycals, NB-600-1235), anti-RIP3
(Santa Cruz, sc-374639), and anti-RIPK1 (Bioss Antibodies, bs-5805R-TR).
The cells were then incubated with secondary antibodies according
to manufacturer’s protocols (Thermo Scientific, TP-125-HL),
followed by incubation with diaminobenzidine (Thermo Scientific, TA-125-HD)
for 5 min to visualize immunolabeling. Following this, the cells were
washed using PBS and counterstained using Mayer’s hematoxylin
(Sigma-Aldrich, 1.07961.0100) for 1 min and mounted using a mounting
medium (Merck Millipore-107961, Germany). All specimens were then
evaluated under a light microscope (Olympus BX40, Tokyo, Japan).
determining the appropriate dose, Vero and HepG2 cells were seeded
for immunocytochemistry analysis, and the
fixed with 4% paraformaldehyde (Merck, 30525) and washed thrice with
phosphate-buffered saline (PBS, Bioshop, PBS404.100). Later, the cells
were treated with Triton X-100 (AppliChem, A4975) on ice for permeabilization
and washed with PBS. Afterward, endogenous peroxidase activity was
quenched by incubation with 3% H2O2 (Merck,
107209) for 5 min at room temperature. The cells were then washed
with PBS and incubated overnight at 4 °C with the following primary
antibodies: anti-Bax (Santa Cruz, sc-526), anti-Bcl2 (Santa Cruz,
sc-7382), anticaspase 3 (Novus Biologycals, NB-600-1235), anti-RIP3
(Santa Cruz, sc-374639), and anti-RIPK1 (Bioss Antibodies, bs-5805R-TR).
The cells were then incubated with secondary antibodies according
to manufacturer’s protocols (Thermo Scientific, TP-125-HL),
followed by incubation with diaminobenzidine (Thermo Scientific, TA-125-HD)
for 5 min to visualize immunolabeling. Following this, the cells were
washed using PBS and counterstained using Mayer’s hematoxylin
(Sigma-Aldrich, 1.07961.0100) for 1 min and mounted using a mounting
medium (Merck Millipore-107961, Germany). All specimens were then
evaluated under a light microscope (Olympus BX40, Tokyo, Japan).
3
Celastrol and LMWH Modulate PI3K/Akt/mTOR Pathway
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Celastrol and low molecular weight heparin (LMWH) were purchased from Dalian Meilun biotechnology company. Dimethylaminopyridine (DMAP), N-Hydroxysuccinimide (NHS) and 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) were obtained from Sigma–Aldrich. Dulbecco's modified Eagle's medium was purchased from Gibco. PI3 Kinase p85α, Phospho-PI3 Kinase p85, Phospho-Akt, anti-Akt, LC3B, P62, mTOR, Phospho-mTOR, and Cleaved Caspase-3 (Asp175) antibodies were purchased from cell signaling technology. Anti-β-actin was purchased from Sigma–Aldrich. Anti-myeloperoxidase was purchased from Abcam. P-selectin and Anti-RIP3 was purchased from Santa Cruz.
4
Immunohistochemical Analysis of RIP3 in Brain
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Consecutive formalin-fixed paraffin-embedded sections of the brains of the animals were subjected to immunostaining as previously described (Zhang et al., 2019 (link)). Briefly, PBS (phosphate buffered saline) containing 1% bovine serum albumin (BSA) was used to block the sections for 1 h, followed by incubation with an anti-RIP3 (Santa Cruz, CA, United States) primary antibody overnight at 4°C. After 3 washes with PBS and 0.1% Tween-20 for 5 min, the sections were stained for 1 h with rhodamine-labeled goat anti-mouse lgG (Abways Technology, Shanghai, China), and the cell nuclei were stained with 4’6-diamidino-2-phenylindole for 5 min. Thereafter, the fluorescence intensity of the as were measured under a Leica DM6B fluorescence microscope (Leica, Wetzlar, Germany).
5
Western Blot Analysis of Key Necroptosis Regulators
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To evaluate the effect of CIS and AZM on RIP1, RIP3, MLKL, and caspase-8, 50 µg protein was subjected to SDS-PAGE followed by transfer onto PVDF membranes. Then, 5% BSA was used for blocking, and the membranes were then incubated with anti-RIP1 (sc-133102—Santa Cruz, Dallas, TX, USA), anti-RIP3 (sc-374639—Santa Cruz, Dallas, TX, USA), anti-MLKL (YPA2507—Biospes, Chongqing, China), anti-caspase-8 (sc-70501—Santa Cruz, USA), and anti-β-actin (sc-8432—Santa Cruz, Dallas, TX, USA) overnight at 4 °C. After washing in TBST, the membranes were incubated with the secondary antibodies for 1 h at RT, washed and visualized using the BCIP/NBT substrate detection kit (GeneMed Biotechnologies, San Francisco, CA, USA). The intensity of the developed bands was determined using ImageJ (NIH, Bethesda, MD, USA).
6
Investigating Necroptosis Signaling Pathways
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Proteins were subjected by SDS/PAGE (12% or 10% gel), and the blots were incubated overnight with primary antibodies. The following primary antibodies were used: anti-RIP1 (Cell Signaling Technology, #3493), anti-RIP3 (Santa Cruz Biotechnology, sc-374639), anti-MLKL (phospho S345) (Abcam, ab196436), anti-MLKL (Cell Signaling Technology, #37705), anti-JNK1+JNK2+JNK3 (Abcam, ab208035), anti-JNK1+JNK2+JNK3 (phospho T183+T183+T221) (Abcam, ab124956), anti-c-Jun (Abcam, ab32137), anti-c-Jun (phospho S73) (Abcam, ab30620), anti-ERK1+ERK2 (Abcam, ab17942), anti-ERK1 (pT202/pY204)+ERK2 (pT185/pY187) (Abcam, ab50011), anti-p38 (Abcam, 170099), anti-p38 (phospho Y182) (Abcam, ab47363), anti-NF-κB (Abcam, ab16502), anti-NF-κB (phospho S536) (Abcam, ab86299), anti-IKBα (Abcam, ab32518), anti-IKBα (phospho S36) (Abcam, ab133462), and anti-GAPDH (Abcam, ab181603).
7
Immunostaining of Necroptosis Pathway
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The discs were fixed by 4% paraformaldehyde (PFA) for 24h at 4°C, followed by dehydration in 25% sucrose solution for 48 h. The tissues were then implanted into optimal cutting temperature compound (Sakura), and serial 12-μm-thick sections were cut on freezing microtome (Leica). All sections were placed on slides and stored at -20°C for immunostaining.
After blocking with 0.01M PBS solution containing 3% BSA and 0.3% Triton X-100 for 30 min, the sections were incubated with primary antibodies in a humid chamber at 4°C overnight. Anti-RIP3(1:300, Santa Cruz Biotechnology, sc-374639), anti-MLKL(1:200, Milipore, MABC 604), anti- p-MLKL(1:200, Abcam, ab187091), and anti-MyD88(1:400, R&D, MAB2928) antibodies were used in this study. The slides were then washed by 0.01M PBS three times, and incubated with corresponding secondary antibodies. The nuclei were stained with Hoechst 33342. The images were photographed by confocal microscope (LSM 800, Zeiss).
After blocking with 0.01M PBS solution containing 3% BSA and 0.3% Triton X-100 for 30 min, the sections were incubated with primary antibodies in a humid chamber at 4°C overnight. Anti-RIP3(1:300, Santa Cruz Biotechnology, sc-374639), anti-MLKL(1:200, Milipore, MABC 604), anti- p-MLKL(1:200, Abcam, ab187091), and anti-MyD88(1:400, R&D, MAB2928) antibodies were used in this study. The slides were then washed by 0.01M PBS three times, and incubated with corresponding secondary antibodies. The nuclei were stained with Hoechst 33342. The images were photographed by confocal microscope (LSM 800, Zeiss).
8
Western Blot Analysis of Protein Markers
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Proteins were extracted as previously described (Sin et al., 2013 (link)). The protein contents were quantified using Bradford Assay. The abundance of interested protein was determined by Western blotting. Briefly, the protein samples were boiled at 95°C in Laemmeli buffer with 5% β-mercaptoethanol, fractioned by SDS-PAGE. They were then transferred onto a PVDF membrane, blocked with 5% milk for an hour, and probed with one of the following primary antibodies at 4°C overnight: anti-SIRT1 (15404, Santa Cruz, Santa Cruz, CA, USA), anti-RIP1 (7881, Santa Cruz), anti-RIP3 (135170, Santa Cruz), anti-NOS2 (650, Santa Cruz), anti-p53 (56179, Santa Cruz), anti-phospho-p53 (101762, Santa Cruz), anti-Bax (493, Santa Cruz), anti-AIF (13116, Santa Cruz), NF-kB p65 (C22B4, Cell Signaling), anti-phospho-p65 NF-kB (Ser536) (3031, Cell signaling), or anti-acetyl-p65 NF-kB (K310) (19870, Abcam). The membranes were washed three times at an interval of 15 min each, probed with the appropriate secondary antibodies, either anti-mouse IgG or anti-rabbit IgG horseradish peroxidase (HRP)-conjugated antibodies (1:4,000; Cell Signaling, Beverly, MA, USA) for 1 h, followed by three washes and the application of luminal reagent (NEL103001EA, Perkin Elmer, Waltham, MA, USA). The signals were captured via BioRad Chemidoc System. Beta-tubulin was used as the internal control and for normalization.
9
Antibody Panel for Signaling Proteins
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Antibodies to detect MET (D1C2), mTOR (7C10), LC3B, PARP, Caspase 3, Cyclin D1, phospho-ALK (Tyr1604), phospho-MET (Tyr1234/1235), AKT, phospho-AKT (Ser473), phospho-MAPK (Tyr202/204), 4EBP1, phospho-4EBP1 (Thr37/46), S6K, phospho-S6K (Ser371), RPS6, phospho-RPS6 (Ser235/236), STAT3, phospho-STAT3 (Tyr705) were from Cell Signaling (Danvers, MA, USA). Anti-RIP3 was from Santa Cruz Biotechnology (Dallas, TX, USA), Anti-Ki-67 from Cell Marque (Rocklin, CA, USA), and Anti-β-Actin from Sigma-Aldrich (München, Germany). ALK-1A4 (immunoblotting) was from Origene (Rockville, MD, USA), ALK-D5F3 (immunohistochemistry) was from Ventana Medical Systems (Tucson, AZ, USA). Crizotinib and rapamycin were purchased from Pfizer (Berlin, Germany) and ChemieTek (Indianapolis, IN, USA), respectively.
10
Immunoprecipitation and Immunoblotting Protocol
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For immunoprecipitation (IP), whole-cell extracts were lysed in an IP buffer composed of 1% (vol/vol) NP-40, 150 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl (pH 7.5) and the complete protease inhibitor cocktail (Roche). Cell lysates were collected and incubated with 1 μg of the corresponding antibodies together with protein G Plus-Agarose Immunoprecipitation reagent (Santa Cruz). After overnight incubation at 4 °C, agaroses were washed five times and boiled with the IP buffer for SDS-PAGE. For immunoblotting analysis, cells were lysed with a lysis buffer (150 mM NaCl, 1 mM EDTA, 1% Triton X-100 and 50 mM Tris-HCl, pH 7.5) supplemented with the protease inhibitor cocktail (Roche), and were incubated with the following antibodies: anti-FLAG (Proteintech, 20543-1-AP), anti-HA (Proteintech, 51064-2-AP), anti-His (Proteintech, 66005-1-lg), anti-RIP1 (Santa Cruz, sc-133102), anti-RIP3 (Santa Cruz, sc-374639), anti-RIP1(Santa Cruz, sc-133102), anti-Myc (Proteintech, 67447-1-Ig), anti-pRIP3 (Abcam, ab205421), anti-mouse pMLKL (Abcam, ab196436), anti-human pMLKL (Abcam, ab206336), anti-MLKL (Abcam, ab243142), anti-Actin (Abcam, ab8226), and anti-TRIM25 (Abcam, ab167154).
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