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).
Anti p mlkl
Manufactured by Abcam
Sourced in United Kingdom, United States
Anti-p-MLKL is a primary antibody that recognizes phosphorylated MLKL (Mixed Lineage Kinase Domain-Like Protein). MLKL is a key mediator of necroptosis, a form of programmed cell death.
Automatically generated - may contain errors
Lab products found in correlation
Anti mlkl, by Abcam (7 mentions)
Anti ripk3, by Abcam (4 mentions)
Anti p rip3, by Abcam (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Goat anti rabbit igg h l, by ZenBio (2 mentions)
Anti gpx4, by Abcam (2 mentions)
Anti p ripk3, by Abcam (2 mentions)
Anti ripk1, by Cell Signaling Technology (2 mentions)
Anti gapdh, by Abcam (2 mentions)
Anti ripk3, by ProSci (2 mentions)
Anti gapdh, by Cell Signaling Technology (2 mentions)
Necrostatin 1, by Merck Group (2 mentions)
Anti caspase 3, by ABclonal (2 mentions)
Anti ripk1, by BD (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
P rip3, by Abcam (1 mentions)
Anti tumour necrosis factor α tnf α, by Abcam (1 mentions)
Anti il 1β, by Abcam (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Alexa fluor 488 goat anti mouse igg secondary antibody, by Thermo Fisher Scientific (1 mentions)
21 protocols using anti p mlkl
1
Protein Expression and Localization in Brain Tissue
2
Western Blotting Protocol for Apoptosis Markers
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RIPA lysis buffer (Beyotime, Nanjing, China) was used to extract proteins from R28 cells. Using BCA Protein Assay Kit (Thermo Fisher Scientific, USA) to quantified the protein concentration. Loaded the protein onto SDS-PAGE and then transferred to PVDF membranes. Subsequently, incubated the membrane using blocking buffer at room temperature. Next, cut the membranes into several blots and incubated it with Anti-Caspase 3 (Abcam, Cambridge, UK, ab184787, 1:2000), Anti-Bax (Abcam, Cambridge, UK, ab32503, 1:5000), Anti-Bcl-2 (Abcam, Cambridge, UK, ab196495, 1:2000), Anti-MLKL (Abcam, Cambridge, UK, ab243142, 1:1000), p-Anti-MLKL (Abcam, Cambridge, UK, ab196436, 1:1000), Anti-Gpx-4 (Abcam, Cambridge, UK, ab125066, 1:1000), Anti-RIP1 (CST, Massachusetts, America, #3493S, 1:1000), Anti-SLC7A11 Polyclonal Antibody (Thermofisher, Waltham, MA, PA1-16893, 1:1000) and β-actin antibody (Sigma-Aldrich, St. Louis, MO, USA, 66009-1-Ig, 1:20,000) at 4 °C overnight. The next day, washed the membranes and incubated it in Goat Anti-Mouse IgG H&L (Zenbio, China, 550017, 1:5000) or Goat Anti-Rabbit IgG H&L (Zenbio, China, 550018, 1:5000) for 90 min. The bands signals were detected using an enhanced chemiluminescence reagent (Bio-Rad) and analyzed using the ImageJ software.
3
Cell Viability, Apoptosis, and Mitochondrial Assays
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Dulbecco's modified Eagle's medium (DMEM) was purchased from Procell (Wuhan, China). Fetal bovine serum (FCS500) was purchased from ExCell Bio (Shanghai, China). Cell counting kit-8 (CCK-8) and Ripa-56 (T7795, 100.00%) were purchased from Topscience (Shanghai, China). Annexin V‐FITC/PI apoptosis detection kit, JC-1 mitochondrial membrane potential assay kit and Hoechst/PI staining buffer were from Beyotime (Shanghai, China). BCA protein assay kit (C05-02001) was purchased from Bioss (Beijing, China). β-actin antibody (66009-1-Ig, 1:20,000) were from Sigma-Aldrich (St. Louis, MO, USA). Anti-Caspase 3 (ab184787), Anti-Bax (ab32503), Anti-Bcl-2 (ab196495), Anti-MLKL (ab243142), p- Anti-MLKL (ab196436), Anti-Brn3a (ab245230, 1:100), Anti-Gpx-4 (ab125066) and Goat Anti-Rabbit IgG H&L (Alexa Fluor 488, ab150077) were obtained from Abcam (Cambridge, UK). Anti-RIP1(#3493S) was purchased from Cell Signaling Technology (CST, Massachusetts, America). Anti-SLC7A11 Polyclonal Antibody (PA1-16893) was from Thermofisher. Anti-IL-6 (EM1701-45, 1:1000) was purchased from HUABIO (Hangzhou, China). Anti RBPMS was from Proteintech (Chicago, America). Goat Anti-Mouse IgG H&L (550017) and Goat Anti-Rabbit IgG H&L (550018) were from Zenbio (China, Chengdu).
4
Histological Analysis of Cecal Tissue
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On day 2 p.i., the cecum was harvested and fixed in 4% paraformaldehyde and then embedded in paraffin; 5 μm sections were used for hematoxylin-eosin (H&E) staining and IHC staining. The assessment of tissue pathology was scored as previously described (Stecher et al., 2005 (link)). For IHC staining, sections were dewaxed and rehydrated, and antigen was unmasked in a citrate-containing buffer, and then immersed the sections in 3% hydrogen peroxide for 10 min, blocked the non-specific antigen with 5% goat serum for 1 h at RT, then incubated with the primary antibody anti-Mucin2 (1:100, Santa Cruz, CA, USA) and anti-p-MLKL (1:100, AbCam, Cambridge, MA, USA) at 4°C overnight respectively, then cover sections with detection antibody.
5
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).
6
Immunofluorescent Staining of p-MLKL in Lm-infected or TNF/CHX-stimulated HepG2 Cells
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HepG2 cells plated on a coverslip were infected with Lm or stimulated with TNF/CHX as described before. Following stimulation, cells were fixed in 4% paraformaldehyde for 10 min at room temperature and thereafter permeabilized with 0.1% Triton-X-100 for 7 min. Nonspecific bindings were blocked by incubating the cells for 45 min in 0.5% BSA, 0.3% Triton-X-100, 5% sucrose, 10% fetal calf serum) before staining the cells with anti-p-MLKL (Cat# ab187091, Abcam, Cambridge, UK) overnight on a rocking platform. The slides were washed three times with PBS and then incubated with the fluorochrome-conjugated secondary antibody (Goat Anti-Rabbit IgG, Alexa Fluor Plus 594, Thermo Fisher Scientific, Waltham, USA) for 2 h protected from light. After washing with PBS, the slides were mounted on a glass slide using ProLong Diamond Antifade with DAPI (Cat# P36962, Thermo Fisher Scientific, Waltham, USA). Images were captured on Microscope Axio Imager Z1 (Carl Zeiss AG, Oberkochen, Germany).
7
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).
8
Immunohistochemical analysis of SARS-CoV-2 infection
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Formalin-fixed paraffin-embedded lung tissue was cut into 4 μm sections and mounted on frosted glass slides. After deparaffinization and rehydration, slides were submerged into pH 8.0 EDTA buffer and boiled for 2 min with high pressure for antigenic retrieval. Slides were washed in PBS, incubated in 3% hydrogen peroxide for 10 min and then blocked with 1% bovine serum albumin (BSA) for 1 h. The following primary antibodies were used: anti-p-hRIPK1 (Cell Signaling Technology, 44590), anti-p-mRIPK1 (BioLynx), anti-RIPK1 (BioLynx, Clone 59), anti-p-MLKL (Abcam, ab187091), anti-p-RIPK3 (Cell Signaling Technology, 93654), anti-CD68 (Zymed, San Francisco, CA) and anti-SARS-CoV/SARS-CoV-2 nucleoprotein (Sino Biological, 40143-T62 and MM05), anti-CD66B (Biolegend, 305102), anti-cleaved Caspase-3 (Cell Signaling Technology, 9661). Slides were then washed in PBST (PBS plus 0.1% Tween 20), incubated with the biotinylated secondary antibody (Zymed, San Francisco, CA) and visualized with 3,3′-diaminobenzidine (DAB) under the microscope.
9
Dot Blot Analysis of Amyloid Fibrils
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Dot blots were done as described previously [48 (link)]. Briefly, 1 μg of protein from the different S1 fractions was first treated with PureProteome Protein G Magnetics Beads (Millipore) for contaminant IgG depletion and then directly applied to dry nitrocellulose, in a final volume of 2 μl. Then, blots were blocked for 1 h and incubated overnight at 4 °C with anti‐Amyloid fibrils (OC, 1:5000, Merck‐Millipore), anti‐prefibrillar oligomers (A11, 1:5000 InvitroGen), anti pMLKL (1:1000, abcam) or anti-GAPDH (1:10,000, Cell Signalling) antibodies. Blots were visualized using the Pierce ECL 2 Western Blotting Substrate detection method (0.5 pg, lower limit sensitivity; Thermo Scientific). The images were obtained and analyzed with ChemiDoc™ Touch Imaging System (Bio‐Rad). Data were always normalized by the specific signal observed in the Braak II group.
10
Molecular Mechanisms of Pentobarbital-Induced Necroptosis
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Pentobarbital sodium was safeguarded in Harbin Medical Pharmacological Laboratory. BSA and rhTrx‐1 were purchased from R&D System. Penicillin/streptomycin solution, Hoechst and 2% TTC dyeing solution were purchased from Solarbio. ROS and Necrostatin‐1 was purchased from Sigma. Annexin V‐PE/7AAD assay was purchased from BD Biosciences. All the ELISA kits were purchased from Cusabio. Dapi dyeing solution and JC‐1 kit were purchased from Beyotime. DMEM, foetal bovine serum (FBS) and MEM/EBSS were purchased from Hyclone. The primary antibodies used for immunofluorescence staining: Rabbit polyclonal anti‐Iba‐1 (Wako), Goat polyclonal anti‐Iba‐1 (Abcam), Mouse monoclonal anti‐RIPK1 (Santa cruz), Goat polyclonal anti‐CD206 (R&D System) and Rabbit polyclonal anti‐CD16 (Bioss). All the second antibodies used for immunofluorescence staining were purchased from Abcam company. The primary antibodies used for Western blot analysis: anti‐RIPK1, anti‐RIPK3, anti‐MLKL, anti‐pMLKL and anti‐CCL2 (Abcam); anti‐NLRP3, anti‐ASC, anti‐caspase‐1, anti‐caspase‐3 and anti‐β‐actin (ABclonal); anti‐MMP‐9 (R&D System); and anti‐CD16 (Bioss). The second antibodies used or Western blot analysis was Alexa Fluor 800‐conjugated Goat‐anti rabbit (LI‐COR).
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