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Ripk1

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RIPK1 is a protein kinase that plays a key role in the regulation of cell death and inflammation pathways. It is involved in the formation of the death-inducing signaling complex (DISC) and the activation of apoptosis and necroptosis. RIPK1 contains a kinase domain and a death domain that are important for its signaling functions.

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Lab products found in correlation

Ripk3, by Santa Cruz Biotechnology (5 mentions) Ecl plus, by Thermo Fisher Scientific (3 mentions) Mitosox red, by Thermo Fisher Scientific (2 mentions) Phospho erk, by Cell Signaling Technology (2 mentions) N galactosamine galn, by Merck Group (2 mentions) Nec 1s 7 cl o nec 1, by Abcam (2 mentions) Nfatc2, by Santa Cruz Biotechnology (2 mentions) Anti gapdh antibody mab374, by Merck Group (2 mentions) Phospho p38α, by Merck Group (2 mentions) Cd4 percp cy5, by Thermo Fisher Scientific (2 mentions) Cd69 pe antibodies, by Thermo Fisher Scientific (2 mentions) Tumor necrosis factor (tnf), by Thermo Fisher Scientific (2 mentions) Hdac1, by Santa Cruz Biotechnology (2 mentions) Anti fas clone jo2, by BD (2 mentions) Cd3 fitc, by BD (2 mentions) Mdivi 1, by Enzo Life Sciences (2 mentions) β actin, by Santa Cruz Biotechnology (2 mentions) Phospho drp1 ser 637, by Cell Signaling Technology (1 mentions) Chloroquine diphosphate, by Merck Group (1 mentions) Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)

13 protocols using ripk1

1

Investigating Cell Death Pathways

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The reagents and sources were as follows: Con A, LPS from Escherichia coli O111:B4, and N-galactosamine (GalN) (Sigma-Aldrich); TNF (Peprotech); α-galactosyl ceramide (α-GalCer, KRN7000) and Mdivi-1 (Enzo Life Sciences); Nec-1s (7-Cl-O-Nec-1, BioVision); FK 506 (LC Labs); cycloheximide (Calbiochem); phospho-ASK1(sc-109911, 1:500 dilution), NFATc2 (sc-7296, 1:1000 dilution), RIPK1 (sc-7881, 1:1000 dilution), RIPK3 (sc-47364, 1:1000 dilution), and HDAC1 (sc-7872, 1:500 dilution) antibodies (Santa Cruz Biotechnology); anti-GAPDH antibody (MAB374, 1:5000 dilution, Chemicon); phospho-p38α (#9211), phospho-JNK (#9251), phospho-ERK (#9101), and phospho-Drp1 (Ser637, #4867) antibodies (1:1000 dilution, Cell Signaling Technology); anti-MLKL antibody (AP14272b, 1:1000 dilution, Abgent); anti-Fas (clone Jo2, #554254, 5 μg per mouse, BD Pharmingen); CD3-FITC (#11-0032, 0.25 μg per 105 cells), CD4-PerCP/Cy5.5 (#35-0042, 0.125 μg per 105 cells), and CD69-PE antibodies (#1-0691, 0.25 μg per 105 cells) (eBioscience); and MitoSOX RED (Molecular Probes). APC-mCD1d/PBS57 ligand tetramers were generously provided by the National Institute of Allergy and Infectious Disease MHC Tetramer Core Facility (Atlanta, GA, USA).
Kang Y.J., Bang B.R., Han K.H., Hong L., Shim E.J., Ma J., Lerner R.A, & Otsuka M. (2015). Regulation of NKT cell-mediated immune responses to tumours and liver inflammation by mitochondrial PGAM5-Drp1 signalling. Nature Communications, 6, 8371.
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2

Autophagy Regulation in Cell Death

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Cell culture materials were obtained from Invitrogen and fetal calf serum was from Gibco. 3-bromopyruvate, chloroquine diphosphate, 3-methyladenine (3-MA), 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), Nec1 were purchased from Sigma-Aldrich. z-VAD-fmk was purchased from Calbiochem. JC-1 and Annexin V-FICT/PI assay were purchased from KeyGEN BioTECH (China). GFP-LC3 plasmid was purchased from GeneCopoeia. The following antibodies were used: LC3, Bclin-1 (MBL), Bax, Bak, Bcl-2, Mcl-1 (ProteinTech), Atg7 (Beyotime), RIPK1 (Santa cruz), RIPK3 (Cell Signaling).
Zhang Q., Zhang Y., Zhang P., Chao Z., Xia F., Jiang C., Zhang X., Jiang Z, & Liu H. (2014). Hexokinase II inhibitor, 3-BrPA induced autophagy by stimulating ROS formation in human breast cancer cells. Genes & Cancer, 5(3-4), 100-112.
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3

Eleutheroside B Inhibits Cisplatin-Induced Injury

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Eleutheroside B was purchased from Chengdu Herbpurify Biotechnology (2018041701, CAS 118-34-3, Chengdu, China). Cisplatin was purchased from Sigma-Aldrich (Sigma, St. Louis, MO, USA). Lactate dehydrogenase (LDH), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), and Annexin V-FITC/PI Apoptosis Detection Kit was obtained from Beyotime (Shanghai, China). Periodic acid-Schiff (PAS), Creatinine Assay Kit, BUN Assay Kit, and Malondialdehyde (MDA) Assay Kit were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and other cell culture reagents were obtained from Invitrogen. Antibodies specific to KIM-1, β-actin, RIPK1, and RIPK3 were purchased from Santa Cruz Biotechnology. Anti-P-MLKL and anti-cleaved-caspase3 were obtained from Cell Signaling Technology (CST, Danvers, MA, USA). IRDye 800-conjugated secondary antibody was purchased from Licor (Lincoln, NE, USA).
Zang H., Yang Q, & Li J. (2019). Eleutheroside B Protects against Acute Kidney Injury by Activating IGF Pathway. Molecules, 24(21), 3876.
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4

Mechanistic Study of Apoptosis Regulation

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The reagents and sources were as follows: Con A, LPS from Escherichia coli O111:B4, and N-galactosamine (GalN) (Sigma-Aldrich); TNF (Peprotech); α-galactosyl ceramide (α-GalCer, KRN7000) and Mdivi-1 (Enzo Life Sciences); Nec-1s (7-Cl-O-Nec-1, BioVision); FK 506 (LC Labs); cycloheximide (Calbiochem); phospho-ASK1(sc-109911, 1:500 dilution), NFATc2 (sc-7296, 1:1,000 dilution), RIPK1 (sc-7881, 1:1,000 dilution), RIPK3 (sc-47364, 1:1,000 dilution), and HDAC1 (sc-7872, 1:500 dilution) antibodies (Santa Cruz Biotechnology); anti-GAPDH antibody (MAB374, 1:5,000 dilution, Chemicon); phospho-p38α (#9211), phospho-JNK (#9251), phospho-ERK (#9101) and phospho-Drp1 (Ser637, #4867) antibodies (1:1,000 dilution, Cell Signaling Technology); anti-MLKL antibody (AP14272b, 1:1,000 dilution, Abgent); anti-Fas (clone Jo2, #554254, 5 μg per mouse, BD Pharmingen); CD3-FITC (#11–0032, 0.25 μg per 105 cells), CD4-PerCP/Cy5.5 (#35–0042, 0.125 μg per 105 cells), and CD69-PE antibodies (#1–0691, 0.25 μg per 105 cells) (eBioscience); and MitoSOX RED (Molecular Probes). APC-mCD1d/PBS57 ligand tetramers were generously provided by the National Institute of Allergy and Infectious Disease MHC Tetramer Core Facility (Atlanta, GA, USA).
Kang Y.J., Bang B.R., Han K.H., Hong L., Shim E.J., Ma J., Lerner R.A, & Otsuka M. (2015). Regulation of NKT cell-mediated immune responses to tumours and liver inflammation by mitochondrial PGAM5-Drp1 signalling. Nature Communications, 6, 8371.
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5

Protein Expression Profiling in Liver Cells

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With 48‐hr treatments of lactoferrin, three MRPs, and the combinations, the HL‐7702 cells were gathered. And the total protein in cells and in liver tissue samples were extracted using a protein extraction kit (Solarbio) and measured by a BCA kit (Solarbio). After 10 min heat treatment of 95℃, the protein samples were added into the 10% SDS–polyacrylamide gels and the electrophoresis was performed, the proteins were transferred onto nitrocellulose membranes by Trans‐Blot machines (Tanon), and the filters were blocked with 2% BSA in TBST buffer for 1.5 hr at room temperature (RT). Then, the proteins were probed with different primary antibodies for 3 hr at RT, including β‐actin, RIPK‐1, RIPK‐3, MLKL, P‐MLKL, TNF‐α, and IL‐1β (Santa Cruz), respectively. The β‐actin was utilized to promise the equal loadings. After washing with PBST buffer (3 × 10 min), the membranes were incubated with the according secondary antibodies for 1.5 hr at RT and then washed with PBST buffer (4 × 15 min). Finally, the membranes were detected utilizing an ECL reagent and analyzed by Image J software (Rawak Software, Inc.).
Fan L., Wang F., Yao Q., Wu H., Wen F., Wang J., Li H, & Zheng N. (2021). Lactoferrin could alleviate liver injury caused by Maillard reaction products with furan ring through regulating necroptosis pathway. Food Science & Nutrition, 9(7), 3449-3459.
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6

Comprehensive Cell Lysate Analysis

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Total Cell Lysate was prepared in RIPA (purchased from Boston Bioproducts, Ashland, MA, USA). Measurement of protein concentration, independent of the extraction method, was performed using BCA assay kit from Pierce, Grand Island, NY, USA. SDS-PAGE and immunoblotting was performed using standard protocol. The cell lysates were separated on 10% or 15% glycine SDS-PAGE gel and transferred to PVDF membrane. Membranes were blocked in 5% non-fat dry milk in TBS with 0.1% TWEEN-20 (TBST) for 1 h at room temperature followed by incubation with indicated primary antibodies in TBST with 5% BSA. Antibodies were purchased from following vendors. BMI-1 was from Invitrogen (37-5400), Bethyl Laboratories Montgomery, TX, USA (IHC-00606) and Proteintech, Rosemont, IL, USA (66161); uH2A (8240), H2A (2578), RING1A (2820), LC3B (2775), β-Actin (4970) , PARP (9542), Cleaved Caspase-3 (9664), Cleaved Caspase-7 (8438), Cleaved Caspase-9 (7237), NFkB/p65 (4764) from Cell Signaling Technology (Danvers, MA, USA); RIPK1 (374639) from Santa Cruz Biotechnology (Dallas, TX, USA); XIAP (MAB822) from R&D Systems (Minneapolis, MN, USA) and secondary antibodies conjugated with horseradish peroxidase IgG Rabbit (A6154) and Mouse (A4416) from Sigma–Aldrich . Primary antibodies were used in dilutions recommended by the manufacturer. Secondary antibodies were used at a concentration of 1:10,000.
Dey A., Xiong X., Crim A., Dwivedi S.K., Mustafi S.B., Mukherjee P., Cao L., Sydorenko N., Baiazitov R., Moon Y.C., Dumble M., Davis T, & Bhattacharya R. (2017). Evaluating the mechanism and therapeutic potential of PTC-028, a novel inhibitor of BMI-1 function in ovarian cancer. Molecular cancer therapeutics, 17(1), 39-49.
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7

Glucocorticoid Receptor Phosphorylation Dynamics

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Cells were seeded with DCC-FBS RPMI media in the presence or absence of CM, treated with Dex/Etop for the indicated times and western blot analysis performed as described previously [36 (link)]. Cells were lysed in high salt lysis buffer and protein concentration was determined using the BioRad assay. Antibodies recognizing actin (Sigma-Aldrich), GR (H300, Santa Cruz Biotechnology), GR phosphorylated at S211 (Abcam), GR phosphorylated at S226 (Abcam), BECN1 (Abcam), RIPK1 (Santa Cruz Biotechnology) and caspase-3 (New England Biolabs) were used. ImageJ was used for quantifying blots [37 (link)].
Qattan M.Y., Bakker E.Y., Rajendran R., Chen D.W., Saha V., Liu J., Zeef L., Schwartz J.M., Mutti L., Demonacos C, & Krstic-Demonacos M. (2017). Differential regulation of cell death pathways by the microenvironment correlates with chemoresistance and survival in leukaemia. PLoS ONE, 12(6), e0178606.
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8

Western Blot Analysis of Cellular Proteins

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Equal amounts of protein were resolved by SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane. Antibodies and dilutions used in this study were as follows: 1:2,000 AIP1/ALIX (Millipore), 1:4,000 RIPK1, 1:7,500 hnRNP K (Santa Cruz), 1:1,000 PFAS (abcam), 1:2,000 ACYL (Millipore), 1:1,000 VDP p115/p115 (Novus Biologicals), 1:2,000 p115 (abcam), 1:1,000 GBF1 (abcam), 1:500 α-tubulin (Santa Cruz), 1:3,000 G3BP1 (BD Transduction Science), 1:3,000 VP1 (Dako), 1:2,000 Flag (Sigma-Aldrich), and 1:1,000 PARP (Santa Cruz).
Jagdeo J.M., Dufour A., Klein T., Solis N., Kleifeld O., Kizhakkedathu J., Luo H., Overall C.M, & Jan E. (2018). N-Terminomics TAILS Identifies Host Cell Substrates of Poliovirus and Coxsackievirus B3 3C Proteinases That Modulate Virus Infection. Journal of Virology, 92(8), e02211-17.
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9

Western Blot Analysis of Cell Signaling

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Protein was extracted using SDS lysis buffer and transferred to PVDF membranes. After the membranes were blocked with 5% skim milk for 1 h, they were incubated with primary antibodies overnight at 4 °C, which was followed by incubation with secondary antibodies for 2 hours. Bands were visualized using a C-Digit Blot Scanner (Gene Company) and analyzed with ImageJ software. GADPH (1:1000, sc-47724, Santa Cruz) was used as a protein loading control. The following primary antibodies were used: antibodies against eIF4E (1:200, sc-9976) from Santa Cruz, RIPK1 (1:500, ab106393), VE-cadherin (1:500, ab33168), vimentin (1:1000, ab92547), and Snail (1:1000, ab180714) from Abcam (Cambridge, USA), phospho-AKT (S473) (1:500, T40067F) from Abmart, AKT (1:500, #AF6259) from Affinity, E-cadherin (1:1000, #3195) from Cell Signaling Technology, and MMP-2 (1:500, 10373-2-AP) from Proteintech.
Li F., Sun H., Yu Y., Che N., Han J., Cheng R., Zhao N., Guo Y., Huang C, & Zhang D. (2023). RIPK1-dependent necroptosis promotes vasculogenic mimicry formation via eIF4E in triple-negative breast cancer. Cell Death & Disease, 14(5), 335.
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10

Modulating Cell Death Pathways

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Commercially available siRNA targeting RIPK1, RIPK3, MLKL, caspase-1, caspase-2, caspase-4, caspase-8 and caspase-10 (Santa Cruz, Dallas, TX) were used to transfect A549 alveolar epithelial cells following the manufacturer instructions. Commercially available CRISPR-Cas9 plasmids to knock out MLKL (Santa Cruz, Dallas, TX) were used following the manufacturer instructions.
Gonzalez-Juarbe N., Bradley K.M., Riegler A.N., Reyes L.F., Brissac T., Park S.S., Restrepo M.I, & Orihuela C.J. (2018). Bacterial Pore-Forming Toxins Promote the Activation of Caspases in Parallel to Necroptosis to Enhance Alarmin Release and Inflammation During Pneumonia. Scientific Reports, 8, 5846.
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