Journal: PLoS ONE

Article Title: RIP1-Dependent and Independent Effects of Necrostatin-1 in Necrosis and T Cell Activation

doi: 10.1371/journal.pone.0023209

Figure Lengend Snippet: (A) Nec-1 inhibits TNF-induced necrosis in L929 cells. L929 cells were treated with 10 µM zVAD-fmk, 20 µM Nec-1 and 10 ng/ml mouse TNF (mTNF) as indicated. Cell death was determined by MTS assay as described in materials and methods. (B) L929 cells were treated with the indicated doses of Nec-1, followed by stimulation with 10 ng/ml mTNF. Cell Death was analyzed as in (A). (C) Nec-1 did not alter RIP1 or RIP3 protein expression in L929 cells.

Article Snippet: After transfer to nitrocellulose membranes, Western blots were performed with antibodies to RIP1, §-actin, PARP-1 (BD Pharmingen, San Diego, CA, USA), RIP3 (ProSci, Poway, CA, USA), Erk, Jnk, PKA substrate (Cell Signaling, Danvers, MA, USA), PKA, LAT (Santa Cruz, Santa Cruz, CA, USA), and phospho-tyrosine (Millipore, Bellerica, MA, USA).

Techniques: MTS Assay, Expressing

Journal: PLoS ONE

Article Title: RIP1-Dependent and Independent Effects of Necrostatin-1 in Necrosis and T Cell Activation

doi: 10.1371/journal.pone.0023209

Figure Lengend Snippet: (A–C) RIP1 is required for zVAD-fmk, but not TNF-induced necrosis in L929 cells. (A) L929 cells were transfected with the indicated siRNA oligonucleotides with HiPerfect (Qiagen). TR4 is the control siRNA against TRAIL-R4 . Cells were treated with (A) 50 µM zVAD-fmk or (B) 10 ng/ml mTNF and cell death was measured by MTS assay. (C) Western blot shows RIP1 and RIP3 expression in cells transfected with the indicated siRNAs. (D–E) RIP1 is required for TNF-induced necrosis in 3T3 fibroblasts. NIH 3T3 fibroblasts were transfected with the indicated siRNA. (D) Expression of RIP1, RIP3 and ß-actin in cells transfected with the indicated siRNA was determined by Western blot. (E) Necrosis was induced with 10 µM zVAD-fmk, 0.5 µg/ml cycloheximide and 10 ng/ml mTNF for 20 hours. Cell death was assessed as in (A).

Article Snippet: After transfer to nitrocellulose membranes, Western blots were performed with antibodies to RIP1, §-actin, PARP-1 (BD Pharmingen, San Diego, CA, USA), RIP3 (ProSci, Poway, CA, USA), Erk, Jnk, PKA substrate (Cell Signaling, Danvers, MA, USA), PKA, LAT (Santa Cruz, Santa Cruz, CA, USA), and phospho-tyrosine (Millipore, Bellerica, MA, USA).

Techniques: Transfection, MTS Assay, Western Blot, Expressing

Journal: BioMed Research International

Article Title: Necroptosis Mediates TNF-Induced Toxicity of Hippocampal Neurons

doi: 10.1155/2014/290182

Figure Lengend Snippet: The regulation of RIP3 in TNF- α -induced toxicity of hippocampal neurons in vivo . (a) Nissl staining of hippocampal neurons 72 h after treatment. Wild-type (WT) and RIP3 knockout (KO) mice received intracerebroventricular injection of PBS or the indicated dose of TNF- α . The neurons of brain sections from WT and KO mice were analyzed by Nissl staining ( n = 7) and morphology of hippocampal CA3 region was shown. Arrows indicate the loss of hippocampal neurons. (b) and (c) Expressions of RIP1, RIP3, and caspase-3 in the hippocampus after TNF - α treatment. Proteins extracted from hippocampus in the wild-type mice treated with PBS or TNF- α were analyzed by western blot using indicated antibodies. PC: MEF cells were treated with staurosporine at 150 nM for 15 hours. The results shown here are representative of five mice.

Article Snippet: The following antibodies were used for western blotting: mouse RIP3 (Prosci, 2283), RIP1 (BD Biosciences, 610459), mouse CYLD (Cell Signaling, 437700), caspase-3 (Cell Signaling, 9662), and β -actin (Sigma).

Techniques: In Vivo, Staining, Knock-Out, Injection, Western Blot

Journal: BioMed Research International

Article Title: Necroptosis Mediates TNF-Induced Toxicity of Hippocampal Neurons

doi: 10.1155/2014/290182

Figure Lengend Snippet: TNF- α -induced necrosis of HT-22 cells depends on RIP3 and its kinase activity. (a) HT-22 cells were transfected with the negative control (NC) or RIP3 siRNAs. After 60 h, cells were treated with control or TNF- α /z-VAD for another 20 h and then cell viability was determined by measuring ATP levels. Data were represented as mean ± standard deviation of duplicates. * P < 0.01, ** P < 0.001 versus NC-T + Z. (b) The knockdown efficiency of RIP3 RNAi. Cell lysates were collected 60 h after transfection and subjected to western blot analysis of RIP3 and β -actin levels. (c) HT-22 cells stably expressing a siRNA-resistant WT-RIP3 or RIP3-K51A or RIP3-RHIM-Mut were transfected with the control or RIP3 siRNAs. After 60 h, cells were treated with control or TNF- α /z-VAD for 20 h and then cell viability was determined by measuring ATP levels. Data were represented as mean ± standard deviation of duplicates. * P < 0.01, ** P < 0.001 versus NC-T + Z. WT-RIP3: HT-22 cells stably expressing a siRNA-resistant wild-type form of RIP3; RIP3-K51A: HT-22 cells stably expressing a siRNA-resistant RIP3 kinase dead mutant. RIP3-RHIM Mut: HT-22 cells stably expressing a siRNA-resistant RHIM domain mutant form of RIP3. (d) The knockdown efficiency of RIP3 RNAi. Cell lysates were collected 60 h after transfection and subjected to western blot analysis of RIP3 and β -actin levels. All experiments were repeated three times with similar results.

Article Snippet: The following antibodies were used for western blotting: mouse RIP3 (Prosci, 2283), RIP1 (BD Biosciences, 610459), mouse CYLD (Cell Signaling, 437700), caspase-3 (Cell Signaling, 9662), and β -actin (Sigma).

Techniques: Activity Assay, Transfection, Negative Control, Standard Deviation, Western Blot, Stable Transfection, Expressing, Mutagenesis

Journal: Science Immunology

Article Title: ZBP1-dependent inflammatory cell death, PANoptosis, and cytokine storm disrupt IFN therapeutic efficacy during coronavirus infection

doi: 10.1126/sciimmunol.abo6294

Figure Lengend Snippet: ( A – C ) Immunoblot analysis of ( A ) pro- (P53) and activated (P30) gasdermin D (GSDMD), pro- (P53) and activated (P34) gasdermin E (GSDME); ( B ) pro- (P55) and cleaved caspase-8 (CASP8; P18), pro- (P35) and cleaved caspase-3 (CASP3; P19 and P17) and pro- (P35) and cleaved caspase-7 (CASP7; P20); and ( C ) phosphorylated MLKL (pMLKL), total MLKL (tMLKL), phosphorylated RIPK3 (pRIPK3) and total RIPK3 (tRIPK3) in the lung samples from mock- or IFN-β–treated wild type (WT) mice with or without mouse hepatitis virus (MHV) infection 3 days post-infection. ( D – I ) Immunoblot analysis of ( D , G ) pro- (P45) and activated (P20) caspase-1 (CASP1), pro- (P53) and activated (P30) GSDMD, pro- (P53) and activated (P34) GSDME; ( E , H ) pro- (P55) and cleaved CASP8 (P18), pro- (P35) and cleaved CASP3 (P19 and P17) and pro- (P35) and cleaved CASP7 (P20); ( F) pMLKL, tMLKL, pRIPK3 and tRIPK3; and ( I ) pRIPK3 and tRIPK3 in mock- or IFN-β–treated bone marrow-derived macrophages (BMDMs) or THP-1 cells during MHV or SARS-CoV-2 infection, respectively. Actin was used as the internal control. Molecular weight marker sizes in kDa are indicated in small font on the left of each blot. Asterisk denotes non-specific bands ( A, G ). Data are representative of at least three independent experiments.

Article Snippet: Following electrophoretic transfer of proteins onto PVDF membranes (Millipore, IPVH00010), non-specific binding was blocked by incubation with 5% skim milk; then membranes were incubated with the following primary antibodies: anti-caspase-1 (AdipoGen, AG-20B-0042, 1:1000), anti-caspase-3 (CST, #9662, 1:1000), anti-cleaved caspase-3 (CST, #9661, 1:1000), anti-caspase-7 (CST, #9492, 1:1000), anti-cleaved caspase-7 (CST, #9491, 1:1000), anti-caspase-8 (CST, #4927, 1:1000), anti-cleaved caspase-8 (CST, #8592, 1:1000), anti-pRIPK3 (CST, #91702S, 1:1000), anti-RIPK3 (ProSci, #2283, 1:1000), anti-pMLKL (CST, #37333, 1:1000), anti-MLKL (Abgent, AP14272b, 1:1000), anti-GSDMD (Abcam, ab209845, 1:1000), anti-GSDME (Abcam, ab215191, 1:1000), anti-pSTAT1 (CST, #7649, 1:1000), anti-STAT1 (CST, #14994, 1:1000), anti-ZBP1 (AdipoGen, AG-20B-0010, 1:1000), anti-β-actin (Proteintech, 66009-1-IG, 1:5000), human anti-caspase-1 (R&D systems; Cat# MAB6215, 1:1000), human anti-caspase-8 (Enzo, ALX-804-242, 1:1000), human anti-GSDMD (Abcam, ab210070, 1:1000), human anti-tMLKL (Abcam, ab184718, 1:1000), human anti-β-actin (CST, 4970, 1:1000), human anti-ZBP1 (R&D systems; Cat# AF6309, 1:1000) and anti-ISG15 (Santa Cruz, sc-166755).

Techniques: Western Blot, Infection, Derivative Assay, Molecular Weight, Marker

Journal: Science Immunology

Article Title: ZBP1-dependent inflammatory cell death, PANoptosis, and cytokine storm disrupt IFN therapeutic efficacy during coronavirus infection

doi: 10.1126/sciimmunol.abo6294

Figure Lengend Snippet: ( A – C ) Immunoblot analysis of ( A ) pro- (P53) and activated (P30) gasdermin D (GSDMD), pro- (P53) and activated (P34) gasdermin E (GSDME); ( B ) pro- (P55) and cleaved caspase-8 (CASP8; P18), pro- (P35) and cleaved caspase-3 (CASP3; P19 and P17) and pro- (P35) and cleaved caspase-7 (CASP7; P20); and ( C ) phosphorylated MLKL (pMLKL), total MLKL (tMLKL), phosphorylated RIPK3 (pRIPK3) and total RIPK3 (tRIPK3) in the lung samples from PBS-treated mice or mouse hepatitis virus (MHV)-infected wild type (WT) and Zbp1 –/– mice treated with IFN-β harvested 3 days after infection. ( D – F ) Immunoblot analysis of ( D ) pro- (P45) and activated (P20) caspase-1 (CASP1), pro- (P53) and activated (P30) GSDMD, pro- (P53) and activated (P34) GSDME; ( E ) pro- (P55) and cleaved (P18) CASP8, pro- (P35) and cleaved (P19 and P17) CASP3 and pro- (P35) and cleaved (P20) CASP7; and ( F ) pMLKL, tMLKL and ZBP1 in PBS- or IFN-β–treated WT, Zbp1 –/– and Zbp1 ∆Za2 bone marrow-derived macrophages (BMDMs) during MHV infection. Actin was used as the internal control. Data are representative of at least three independent experiments.

Article Snippet: Following electrophoretic transfer of proteins onto PVDF membranes (Millipore, IPVH00010), non-specific binding was blocked by incubation with 5% skim milk; then membranes were incubated with the following primary antibodies: anti-caspase-1 (AdipoGen, AG-20B-0042, 1:1000), anti-caspase-3 (CST, #9662, 1:1000), anti-cleaved caspase-3 (CST, #9661, 1:1000), anti-caspase-7 (CST, #9492, 1:1000), anti-cleaved caspase-7 (CST, #9491, 1:1000), anti-caspase-8 (CST, #4927, 1:1000), anti-cleaved caspase-8 (CST, #8592, 1:1000), anti-pRIPK3 (CST, #91702S, 1:1000), anti-RIPK3 (ProSci, #2283, 1:1000), anti-pMLKL (CST, #37333, 1:1000), anti-MLKL (Abgent, AP14272b, 1:1000), anti-GSDMD (Abcam, ab209845, 1:1000), anti-GSDME (Abcam, ab215191, 1:1000), anti-pSTAT1 (CST, #7649, 1:1000), anti-STAT1 (CST, #14994, 1:1000), anti-ZBP1 (AdipoGen, AG-20B-0010, 1:1000), anti-β-actin (Proteintech, 66009-1-IG, 1:5000), human anti-caspase-1 (R&D systems; Cat# MAB6215, 1:1000), human anti-caspase-8 (Enzo, ALX-804-242, 1:1000), human anti-GSDMD (Abcam, ab210070, 1:1000), human anti-tMLKL (Abcam, ab184718, 1:1000), human anti-β-actin (CST, 4970, 1:1000), human anti-ZBP1 (R&D systems; Cat# AF6309, 1:1000) and anti-ISG15 (Santa Cruz, sc-166755).

Techniques: Western Blot, Infection, Derivative Assay

Journal: Cell Death & Disease

Article Title: The Lck inhibitor, AMG-47a, blocks necroptosis and implicates RIPK1 in signalling downstream of MLKL

doi: 10.1038/s41419-022-04740-w

Figure Lengend Snippet: A , B U937 cell lines of various genotypes expressing an inducible MLKL(1-180)-gyrase fusion were treated with doxycycline (to induce expression) and/or coumermycin (to induce dimerisation), or left untreated. After 24 h ( A ) or 48 h ( B ), cells were harvested and assessed for cell death (PI uptake measured using flow cytometry), counting a minimum of 5000 cells. Data represent three independent experiments, with the exception of the BAX −/− BAK −/− cell lines, where n = 2. Statistics were calculated in GraphPad Prism 8, and p values are shown where <0.05. C The same U937 cells used in panel A and B were treated with doxycycline alone and left overnight. After approximately 16 h, cells were harvested and lysed for analysis of fusion protein expression via western blot. The upper MLKL band (*) represents endogenous MLKL, and the lower band (arrow) represents the MLKL (1-180) -gyrase fusion protein. D Wild-type U937 were treated with TNF (100 ng/ml), Smac-mimetic (0.5 µM), and caspase inhibitor IDN-6556 (emricasan; 5 µM) (TSI) to induce necroptosis or TNF and Smac-mimetic (TS) alone to induce apoptosis. In parallel, MLKL −/− U937 expressing an inducible MLKL (1-180) -gyrase fusion were treated with doxycycline and coumermycin in the presence or absence of necrosulfonamide (NSA). Images were taken at the specified timepoints using an IncuCyte S3 System to track morphological changes and PI uptake (shown by red). Cells with a necroptotic phenotype (cellular swelling) are indicate with yellow arrows. Three fields were examined per well with representative data shown. E Wild-type U937 cells were treated with doxycycline (20 nM) to induce expression of human wild-type RIPK3 in the presence of various cell death inhibitors, as indicated, for 24 h. At the conclusion of the experiment, cells were analysed for PI uptake using flow cytometry, with a minimum of 5000 cells counted. Data represent four or more independent experiments. F Wild-type, MLKL −/− and RIPK1 −/− U937 expressing inducible human wild-type RIPK3 were treated with doxycycline (20 nM) and IDN-6556 (5 µM), doxycycline alone, or left untreated. After 24 h, cell death was measured as PI uptake using flow cytometry. Data are a summary of six independent experiments.

Article Snippet: The following primary antibodies were used in this study: rat anti-MLKL clone 3H1 (available as MABC604 from Merck Millipore; 1:1000) was produced in-house as previously described [ ]; rabbit anti-human MLKL pS358 (ab187091, Abcam, Cambridge, United Kingdom; 1:4000); rabbit anti-mouse MLKL pS345 (ab196436, Abcam; 1:1000); rat anti-human RIPK3 (clone 1H2; available as MABC1640 from Merck Millipore; 1:1000) and rat anti-mouse RIPK3 (clone 8G7; available as MABC1595 from Merck Millipore; 1:1000) produced in-house as previously described [ , ]; rabbit anti-RIPK3 (2283, ProSci; 1:1000); rabbit anti-human RIPK3 pS227 (ab209384, Abcam; 1:2000); rabbit anti-mouse RIPK3 pT231/pS232 (GEN135-35-9, kindly supplied by Genentech, South San Francisco, CA, USA [ ]; 1:2000); mouse anti-RIPK1 (610458, BD Transduction Laboratories, San Jose, CA, USA; 1:1000); rabbit anti-RIPK1 (3493, Cell Signalling Technology, Danvers, MA, USA; 1:1000); rabbit anti-human RIPK1 pS166 (65746, Cell Signalling Technology; 1:1000); rabbit anti-mouse RIPK1 pS166 (31122, Cell Signalling Technology; 1:1000); rabbit anti-mouse CASP8 (4927, Cell Signalling Technology; 1:1000); rabbit anti-BAX (2772, Cell Signalling Technology; 1:1000); rabbit anti-BAX (06-536, Upstate Biotechnology, Lake Placid, NY, USA; 1:1000); rabbit anti-GAPDH (2118, Cell Signalling Technology; 1:1000); and mouse anti-β-Actin (A1978, Sigma-Aldrich; 1:3000).

Techniques: Expressing, Flow Cytometry, Western Blot

Journal: Cell Death & Disease

Article Title: The Lck inhibitor, AMG-47a, blocks necroptosis and implicates RIPK1 in signalling downstream of MLKL

doi: 10.1038/s41419-022-04740-w

Figure Lengend Snippet: A Three different experimental protocols were used to assess the ability of AMG-47a to inhibit cell death caused by the expression and dimerisation of the MLKL (1-180) -gyrase fusion protein. In protocol 1, inhibitors (or DMSO) were added first, followed by doxycycline and coumermycin together, so that the fusion protein could dimerise on expression. In protocol 2, addition of coumermycin was delayed to allow levels of the fusion protein to accumulate before dimerisation was induced. In protocol 3, the addition of inhibitors (or DMSO) was also delayed, so that it was added after the fusion protein had been expressed for 16 h, but before the addition of coumermycin. In all experimental methods, cells were harvested 24 h after the experiment was initiated. B Wild-type U937 cells expressing the MLKL (1-180) -gyrase fusion protein were treated using either protocol 1, 2, or 3, as described above ( A ). At the conclusion of the experiment, cells were analysed for PI uptake using flow cytometry, with a minimum of 5000 cells counted. Data represent three independent experiments; bars indicate the mean and error bars indicate standard error of the mean. Statistics were calculated in GraphPad Prism8, and p values are shown where <0.05. C Wild-type U937 cells with inducible human RIPK3 were treated with increasing doses of AMG-47a in the presence of 40 nM doxycycline, to induce expression, and 5 µM IDN-6556, to block caspase-dependent cell death. After 48 h, cells were harvested and viability assessed using the Cell-Titer Glo 2 (Promega) system. Data represent two independent experiments (four replicates per experiment) and have been normalised against doxycycline plus IDN-6556 (0% viability) and doxycycline plus IDN-6556 plus NSA (100% viability).

Article Snippet: The following primary antibodies were used in this study: rat anti-MLKL clone 3H1 (available as MABC604 from Merck Millipore; 1:1000) was produced in-house as previously described [ ]; rabbit anti-human MLKL pS358 (ab187091, Abcam, Cambridge, United Kingdom; 1:4000); rabbit anti-mouse MLKL pS345 (ab196436, Abcam; 1:1000); rat anti-human RIPK3 (clone 1H2; available as MABC1640 from Merck Millipore; 1:1000) and rat anti-mouse RIPK3 (clone 8G7; available as MABC1595 from Merck Millipore; 1:1000) produced in-house as previously described [ , ]; rabbit anti-RIPK3 (2283, ProSci; 1:1000); rabbit anti-human RIPK3 pS227 (ab209384, Abcam; 1:2000); rabbit anti-mouse RIPK3 pT231/pS232 (GEN135-35-9, kindly supplied by Genentech, South San Francisco, CA, USA [ ]; 1:2000); mouse anti-RIPK1 (610458, BD Transduction Laboratories, San Jose, CA, USA; 1:1000); rabbit anti-RIPK1 (3493, Cell Signalling Technology, Danvers, MA, USA; 1:1000); rabbit anti-human RIPK1 pS166 (65746, Cell Signalling Technology; 1:1000); rabbit anti-mouse RIPK1 pS166 (31122, Cell Signalling Technology; 1:1000); rabbit anti-mouse CASP8 (4927, Cell Signalling Technology; 1:1000); rabbit anti-BAX (2772, Cell Signalling Technology; 1:1000); rabbit anti-BAX (06-536, Upstate Biotechnology, Lake Placid, NY, USA; 1:1000); rabbit anti-GAPDH (2118, Cell Signalling Technology; 1:1000); and mouse anti-β-Actin (A1978, Sigma-Aldrich; 1:3000).

Techniques: Expressing, Flow Cytometry, Blocking Assay

Journal: Cell Death & Disease

Article Title: The Lck inhibitor, AMG-47a, blocks necroptosis and implicates RIPK1 in signalling downstream of MLKL

doi: 10.1038/s41419-022-04740-w

Figure Lengend Snippet: A, B, C U937, HT29 and MDF cell lines were treated with DMSO, AMG-47a (1 µM for U937 and MDF; 2 µM for HT29), TSI, or a combination of AMG-47a and TSI over a time course as indicated in the individual figure panels. At the conclusion of the experiment, cells were harvested and lysed for western blot analysis. All cell lines were assessed for phosphorylation and total protein of the three key necroptosis effector proteins, RIPK1, RIPK3, and MLKL, and total β-actin was used as a loading control. Where multiple bands are present, the specific band of interest is indicated with a red arrow. Data are representative of at least three independent experiments. U937 ( D ) or MDF ( E ) cell lines were treated with AMG-47a at 10 µM, the RIPK1 inhibitors (GSK-481, Nec-1s) or a RIPK3 inhibitor (GSK-872) at 20 µM, or an equivalent amount of DMSO, for 1 h at 37 °C. Cell suspensions were then heated over an increasing temperature gradient (as indicated in the methods) for 3 min. Cells were then lysed, and the amount of remaining soluble protein at each temperature point was analysed using Western blot. Results are representative of two (RIPK1 and RIPK3 in U937) or three (all other analyses) independent experiments. AMG-47a binding to RIPK1 ( F ) and RIPK3 ( G ) was measured in competitive binding assays using the KINOMEscan ® Assay Platform (DiscoverX). Graphs were plotted using the raw data supplied from DiscoverX, and a curve was fitted using non-linear regression (GraphPad Prism) to determine the K d and associated error (95% confidence interval). Data represent one (RIPK3) or two (RIPK1) independent experiments, with two runs performed in all experiments and each colour on the graph representing a separate run. All data points were used for the non-linear regression. K d determinations for individual replicates can be found in supplementary data (Supplementary Fig. ). H , I AMG-47a was serially diluted from a maximum concentration of 50 µM (RIPK1) or 100 µM (RIPK3), and its ability to inhibit human RIPK1 or RIPK3 kinase activity was assessed using the ADP-Glo Kinase Assay (Promega). The reaction was allowed to proceed for 4 h before readouts were performed, and data were normalised to DMSO (0% inhibition) and either 1 µM GSK-481 (100% inhibition of RIPK1) or 1 µM GSK-872 (100% inhibition of RIPK3). Data are a summary of two independent experiments performed in duplicate, and the IC 50 and associated error (95% confidence interval) were determined using non-linear regression, as above. Each colour on the graph represents one independent experiment.

Article Snippet: The following primary antibodies were used in this study: rat anti-MLKL clone 3H1 (available as MABC604 from Merck Millipore; 1:1000) was produced in-house as previously described [ ]; rabbit anti-human MLKL pS358 (ab187091, Abcam, Cambridge, United Kingdom; 1:4000); rabbit anti-mouse MLKL pS345 (ab196436, Abcam; 1:1000); rat anti-human RIPK3 (clone 1H2; available as MABC1640 from Merck Millipore; 1:1000) and rat anti-mouse RIPK3 (clone 8G7; available as MABC1595 from Merck Millipore; 1:1000) produced in-house as previously described [ , ]; rabbit anti-RIPK3 (2283, ProSci; 1:1000); rabbit anti-human RIPK3 pS227 (ab209384, Abcam; 1:2000); rabbit anti-mouse RIPK3 pT231/pS232 (GEN135-35-9, kindly supplied by Genentech, South San Francisco, CA, USA [ ]; 1:2000); mouse anti-RIPK1 (610458, BD Transduction Laboratories, San Jose, CA, USA; 1:1000); rabbit anti-RIPK1 (3493, Cell Signalling Technology, Danvers, MA, USA; 1:1000); rabbit anti-human RIPK1 pS166 (65746, Cell Signalling Technology; 1:1000); rabbit anti-mouse RIPK1 pS166 (31122, Cell Signalling Technology; 1:1000); rabbit anti-mouse CASP8 (4927, Cell Signalling Technology; 1:1000); rabbit anti-BAX (2772, Cell Signalling Technology; 1:1000); rabbit anti-BAX (06-536, Upstate Biotechnology, Lake Placid, NY, USA; 1:1000); rabbit anti-GAPDH (2118, Cell Signalling Technology; 1:1000); and mouse anti-β-Actin (A1978, Sigma-Aldrich; 1:3000).

Techniques: Western Blot, Binding Assay, Concentration Assay, Activity Assay, Kinase Assay, Inhibition

Journal: Cell Death & Disease

Article Title: The Lck inhibitor, AMG-47a, blocks necroptosis and implicates RIPK1 in signalling downstream of MLKL

doi: 10.1038/s41419-022-04740-w

Figure Lengend Snippet: A The cell death induced by forced dimerisation of the human MLKL NTD involves two distinct processes. In the first, MLKL can self-associate into discrete oligomers on addition of doxycycline. In the second, addition of coumermycin promotes the assembly of large multimeric complexes. AMG-47a is unable to inhibit the second process, thus we propose it prevents membrane attachment, translocation, or oligomerisation of the human MLKL NTD through its activity as a kinase inhibitor. As AMG-47a binds RIPK1, and as RIPK1 is required for this form of cell death, we propose the primary target of AMG-47a is RIPK1 in this context. B When a cell receives a necroptotic stimulus, a number of key events occur, beginning with assembly of a necrosome containing RIPK1, RIPK3, and MLKL. This triggers a number of events, beginning with the phosphorylation of MLKL by RIPK3, which enables a conformational change in MLKL that allows oligomerisation, translocation to cellular membranes, and ultimately cell death. Our data suggest an additional role for RIPK1 in human necroptosis by supporting the conformational change, oligomerisation, or membrane translocation of MLKL following phosphorylation by RIPK3.

Article Snippet: The following primary antibodies were used in this study: rat anti-MLKL clone 3H1 (available as MABC604 from Merck Millipore; 1:1000) was produced in-house as previously described [ ]; rabbit anti-human MLKL pS358 (ab187091, Abcam, Cambridge, United Kingdom; 1:4000); rabbit anti-mouse MLKL pS345 (ab196436, Abcam; 1:1000); rat anti-human RIPK3 (clone 1H2; available as MABC1640 from Merck Millipore; 1:1000) and rat anti-mouse RIPK3 (clone 8G7; available as MABC1595 from Merck Millipore; 1:1000) produced in-house as previously described [ , ]; rabbit anti-RIPK3 (2283, ProSci; 1:1000); rabbit anti-human RIPK3 pS227 (ab209384, Abcam; 1:2000); rabbit anti-mouse RIPK3 pT231/pS232 (GEN135-35-9, kindly supplied by Genentech, South San Francisco, CA, USA [ ]; 1:2000); mouse anti-RIPK1 (610458, BD Transduction Laboratories, San Jose, CA, USA; 1:1000); rabbit anti-RIPK1 (3493, Cell Signalling Technology, Danvers, MA, USA; 1:1000); rabbit anti-human RIPK1 pS166 (65746, Cell Signalling Technology; 1:1000); rabbit anti-mouse RIPK1 pS166 (31122, Cell Signalling Technology; 1:1000); rabbit anti-mouse CASP8 (4927, Cell Signalling Technology; 1:1000); rabbit anti-BAX (2772, Cell Signalling Technology; 1:1000); rabbit anti-BAX (06-536, Upstate Biotechnology, Lake Placid, NY, USA; 1:1000); rabbit anti-GAPDH (2118, Cell Signalling Technology; 1:1000); and mouse anti-β-Actin (A1978, Sigma-Aldrich; 1:3000).

Techniques: Translocation Assay, Activity Assay