Dulbecco's Modified Eagle Medium (DMEM) was purchased from Gibco (Life Technologies, Carlsbad, CA), recombinant mouse TNFα from R&D Systems (Minneapolis, MN), Z-VAD-FMK (zVAD) from Bachem (Torrance, CA), and 7-Cl-O-Nec-1 (Necrostatin-1s, Nec-1s) from EMD Millipore (Burlington, MA). Primary antibodies including anti-MLKL, anti-MLKL (phospho S345, ab196436), anti-SM-αActin, and anti-CD68 were purchased from Abcam (Cambridge, MA), anti-RIP1 was purchased from BD Biosciences (San Jose, CA), anti-RIP3 from ProSci (Poway, CA), anti-β-Actin (ACTB) from Sigma-Aldrich (St. Louis, MO). Fluorophore-conjugated secondary antibodies and 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) were purchased from Molecular Probes (Life Technologies, Carlsbad, CA). Horseradish Peroxidase (HRP)-conjugated antibodies were purchased from Bio-Rad (Hercules, CA). In situ Cell Death Detection Kit was purchased from Roche Applied Science (Indianapolis, IN). Other chemicals and reagents if not specified were purchased from Sigma-Aldrich (St. Louis, MO).
Anti mlkl
Manufactured by Abcam
Sourced in United Kingdom, United States
Anti-MLKL is a primary antibody that recognizes the MLKL (Mixed Lineage Kinase Domain-Like Protein) protein. MLKL is a key mediator of necroptosis, a form of programmed cell death. This antibody can be used to detect and study MLKL expression and its role in cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Anti p mlkl, by Abcam (7 mentions)
Anti ripk1, by Abcam (4 mentions)
Anti ripk3, by Abcam (4 mentions)
Anti rip1, by BD (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Anti phospho mlkl, by Abcam (2 mentions)
Anti β actin, by Abcam (2 mentions)
Horseradish peroxidase, by Santa Cruz Biotechnology (2 mentions)
β actin, by Merck Group (2 mentions)
Anti β actin, by Merck Group (2 mentions)
Anti p ripk3, by Abcam (2 mentions)
Anti rip3, by Cell Signaling Technology (2 mentions)
Smac mimetic lcl 161, by Adooq (2 mentions)
Anti p erk, by Cell Signaling Technology (2 mentions)
Anti caspase 8, by Cell Signaling Technology (2 mentions)
Anti parp, by Cell Signaling Technology (2 mentions)
Tnf α, by R&D Systems (2 mentions)
Anti camkiiδ, by GeneTex (2 mentions)
Anti caspase 3, by ABclonal (2 mentions)
Necrostatin 1, by Merck Group (2 mentions)
23 protocols using anti mlkl
1
Investigating Cell Death Pathways
2
Western Blot Analysis of Cell Death Signaling
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Retina tissue and cells were harvested and lysed in RIPA buffer containing 10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% sodium dodecyl sulfate (SDS), 1% Nonidet P-40, and 1% sodium deoxycholate, and was supplemented with protease and phosphatase inhibitor mini tablets (Thermo Fisher Scientific, Waltham, MA). The protein concentration was determined with bicinchoninic acid protein assay. Western blotting was performed as previously described [23 (link)]. Briefly, 30 μg of protein was loaded per lane on a 10% SDS-PAGE. The membrane blots were saturated with 5% BSA in PBST for 1 h at room temperature and then incubated overnight at 4 °C with antibodies. Primary antibodies used included anti-RIP1 (BD Bio-Sciences, 1:1000), anti-RIP3 (Abcam, 1:1000), anti-phospho-RIP1 (Invitrogen, 1:1000), anti-phospho-RIP3 (Abcam, 1:1000), anti-MLKL (Abcam, 1:1000), anti-phospho-MLKL (Abcam, 1:1000), and anti-β-actin (Abcam, 1:2000) antibodies. Band intensities were measured using the Image J software (US National Institutes of Health). Co-immunoprecipitation assays were performed using the Thermo Scientific Pierce co-IP kit according to the manufacturer’s instruction. The levels of TNF-α and MCP-1 in cultured microglia was detected with ELISA kits (R&D Systems, Minneapolis, MN) according to manufacturer’s instruction.
3
Immunoprecipitation and Immunoblotting of RIP3 and MLKL
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100 μg of whole cell extracts were immunoprecipitated using a rabbit polyclonal anti-RIP3/RIP3K (Abcam Inc., Cambridge, UK), which was diluted 1:100. The samples were agitated overnight at 4 °C. Protein A/G beads (Pierce Inc., Vallejo, CA, USA) were added and the samples were agitated for two more hours at 4 °C and centrifuged at 14,000 rpm for 5 min at 4 °C. The resulting pellet was washed three times, suspended in sample buffer, and boiled for 5 min. The samples were centrifuged at 14,000 rpm for 5 min at room temperature and the resulting supernatants were subjected to immunoblotting, as described above, with mouse monoclonal anti-MLKL (Abcam Inc., Cambridge, UK) and rabbit polyclonal anti-RIP3/RIP3K (Abcam Inc., Cambridge, UK), which served as loading control.
4
Neutrophil Necroptosis Signaling Assay
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S. aureus supernatant was obtained by centrifugation and filtration. HKLAC was obtained by incubating LAC cells in PBS (1 × 109 CFU/mL) at 65 °C for 2 h to inactivate the bacteria. Neutrophil lysates were run on bolt 4–12% Bis-Tris Plus gels (Life Technologies) and transferred to polyvinylidene difluoride membranes (Millipore). Then the membrane was blocked with 5% milk in TBST (Tris-buffered saline plus Tween) for 1.5 h at room temperature. Immunodetection was performed using anti-phospho-MLKL (Ser358) (Abcam, Cambridge, UK), anti-MLKL (Abcam, Cambridge, UK), anti-TNFα (R&D Systems, USA), and β-actin (Sigma Aldrich Chemical Co, USA) antibodies followed by secondary antibodies conjugated to horseradish peroxidase (Santa Cruz Biotechnology Inc, USA).
5
Detecting RIPK3 and MLKL Oligomers
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Western blotting experiment was described previously (Zhao et al., 2019 (link)), and the detection of RIPK3 or MLKL oligomers was performed according to the method described by Wang et al. (2019) (link). The following antibodies were used for the experiments: anti-FADD (1:1000, 610399) and anti-RIPK1 (1:3000, 610458) (BD Transduction Laboratories, San Jose, CA); anti-phospho-RIPK3 (1:1000, ab195117), anti-MLKL (1:1000, ab172868), anti-phospho-MLKL (1:1000, ab196436) (Abcam, Cambridge, MA, United States); anti-RIPK1 (1:3000, 3493), anti-RIPK3 (1:3000, 15828), anti-Myc tag (1:2000, 2276), anti-HA tag (1:2000, 3724), anti-Flag tag (1:2000, 14793), anti-TRAF2 (1:1000, 4724), anti-phospho-RIPK1 (1:1500, 31122) and anti-phospho-RIPK3 (1:1000, 57220) [Cell Signaling Technology (CST), Beverly, MA]; anti-TRADD (1:1000, ABP52634) and anti-GAPDH (1:1500, ABP52783) (Abbkine, Redlands, CA, United States); anti-β-actin (1:3000, A5441) (Sigma-Aldrich). All western blot assays were performed three times, and the representative results are shown.
6
Protein Expression Analysis in Lung Tissue
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A suitable amount of lung tissue stored in -80°C freezer was homogenated and lysed in RIPA buffer containing proteinase and phosphatase inhibitor cocktail (CWBIO Co. Ltd., China). After protein concentrations were assessed using the BCA protein assay kit (CWBIO Co. Ltd., China), protein samples were boiled in loading buffer for 10 min. 40 μg protein samples were then run on a 10% SDS-PAGE and transferred onto a PVDF membrane (Millipore, USA) at 250mA for 2h. The membrane was blocked with 5% fat-free milk in TBST (0.1% Tween-20 in TBS) for 2h at room temperature and incubated with primary antibodies at 4°C over night. Primary antibodies used in this study were anti-cIAP2 antibody(1:1000, R&D system), anti-RIPK1 antibody (1:1000, Novus), anti-RIPK3 antibody (1:1000, Abcam), anti-MLKL (1:1000, Abcam), anti-RIP3 (phospho S227) antibody (1:2000, Abcam), anti-MLKL (phospho S345) antibody (1:1000, Abcam), and anti-GAPDH antibody(1:4000, CWBIO Co. Ltd). Then the membranes were washed 3 times in TBST and incubated with goat-anti-mouse/rabbit antibody for 2h at room temperature. After being washed 3 times in TBST again, the protein bands were visualized by fluorography using ECL (enhanced chemiluminescence) reagents (Millipore, USA) and analyzed by image J.
7
Western Blot Analysis of BEC Proteins
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Confluent BECs in 6-well plates were exposed to in vitro grown VTP (3 × 107 cells/well) or equally diluted IEC for 5 h with 3 biological replicates per treatment condition. Cells were lysed for 30 min on ice with gentle agitation in RIPA lysis buffer containing EDTA-free Protease inhibitor cocktail set 3 (Calbiochem, San Diego, CA) and PhosSTOP phosphatase inhibitor (Roche, Mississauga, ON) according to manufacturer’s instructions. Protein concentration was measured using a Pierce BCA assay (Thermo Fisher Scientific). For western blotting, the 3 biological replicates of BEC lysate from each treatment group (VTP- or IEC-exposed) were pooled, and 13.5 μg pooled whole cell lysate per treatment group was subjected to SDS-PAGE using Bolt 12% acrylamide gels (Thermo Fisher Scientific) and transferred to PVDF membrane (Millipore) via wet-transfer at 400 mA for 2 h, and blocked with Intercept TBS blocking buffer (Licor, Lincoln, NE). Anti-fibronectin (1:1,000) (Sigma-Aldrich, F-3648), anti-MLKL (1:1,000) (Abcam, pS358 EPR9514), and anti-GAPDH (1:1,000) (Cell Signaling Technology, D4C6R) were used as primary antibodies, while the secondary antibodies used were goat anti-rabbit IgG IRDye 800CW (1:20,000) and goat anti-mouse IgG IRDye 680RD (1:20,000) (Licor). Detection and analysis were completed on a Licor Odyssey CLx using Licor Image Studio version 5.2.
8
Adipose Tissue Protein Extraction and Western Blot Analysis
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Total proteins of adipose tissues were extracted and western blot was carried out as previously described51 (link). The following antibodies were used: anti-β-Actin (1:1000, Protein Tech, 60008-1-Ig), anti-MED20 (1;1000, Protein Tech, 17598-1-AP), anti-GAPDH (1:5000, CST, 5174 s), anti-MLKL (1:1000, Abcam, ab184718), anti-P-MLKL (1:1000; Abcam, ab187091), anti-Cleaved Caspase-3 (1:1000, CST, 4190), anti- Acetyl-CoA Carboxylase1 (1:1000, CST, 4190), anti-ACLY (1:1000, CST, 13390), anti- RIPK3 (1:1000, ABclonal, A5431), anti-RIPK1 (1:1000, ABclonal, A7414), anti-P-RIPK3 (1:1000, Abcam, ab205421), anti-P-RIPK1 (1:1000, ABclonal, AP1230), anti-C/EBPα (1:1000, CST, 8178 S), anti-PPARγ (1:1000, Santa Cruz, sc-7273), anti-CD36 (1:1000, Sino Biological Inc, 80263-T48), anti-Perilipin 1 (1:1000, CST, 9349 s) and anti-FASN(1:1000, CST, 3180).
9
Antibodies for Immunoblotting and Immunofluorescence
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Antibodies used in immunoblotting and immunofluorescence: anti-MLKL (Abcam, ab184718, 1:2000), anti-RIP3 (Cell Signaling Technology, 13526, 1:1000), anti-ACTIN (Santa Cruz Biotechnology, 47778, 1:5000), anti-VINCULIN (Sigma-Aldrich, V9131, 1:5000), anti-PARP (Cell Signaling Technology, 9542, 1:1000), anti-AP2a (BD biosciences, 610501, 1:1000), anti-Caspase-3 (Cell Signaling Technology, 9662, 1:1000), anti-Caspase-8 (Cell Signaling Technology, 9746, 1:1000), anti-FADD (BD biosciences, 610400, 1:1000), anti-DR5 (Abcam, ab199357, 1:1000), anti-EGFR (Abcam, ab2430, 1:1000), anti-p-ERK (Cell Signaling Technology, 9101, 1:1000), anti-p-AKT (Cell Signaling Technology, 9271, 1:1000), anti-p-p38 (Cell Signaling Technology, 9215, 1:1000) and anti-GST (Abcam, ab9085, 1:500). TNF-α and zVAD were purchased from R&D Systems. Etoposide and Dynasore were purchased from Sigma-Aldrich. SMAC mimetic (LCL-161) was purchased from Adooq Bioscience. 4 hydroxytamoxifen was purchased from Sigma-Aldrich.
10
Quantitative Immunofluorescence Analysis
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Cells on coverslips were blocked (1 × PBS, 2% normal goat serum, and 0.1% Triton X-100) for 1 h and incubated overnight at 4 °C with the following primary antibodies: anti-RIPK1 (1:400; Abcam, Burlingame, CA, USA), anti-RIPK3 (1:400, Abcam), anti-MLKL (1:400, Abcam), and anti-CaMKIIδ (1:100; GeneTex, Irvine, CA, USA). Cells were washed three times with 0.1 M PBS and then incubated with Cy3-labeled secondary antibody (1:400; Beyotime, Shanghai, China) for 1 h at room temperature. The cells were then photographed under a fluorescent microscope (Leica, Microsystems, Wetzlar, Germany) with an excitation wavelength of 550 nm and an emission wavelength of 570 nm.
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