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Ac yvad cmk

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Ac-YVAD-CMK is a synthetic peptide that functions as a caspase-1 inhibitor. It is commonly used in research applications to study the role of caspase-1 in cellular processes.

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Lab products found in correlation

Lipopolysaccharide lps from escherichia coli 0111 b4, by Merck Group (2 mentions) Methamphetamine, by Merck Group (2 mentions) Nigericin, by Merck Group (2 mentions) Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions) Penicillin, by Thermo Fisher Scientific (1 mentions) Streptomycin, by Thermo Fisher Scientific (1 mentions) Adenosine triphosphate (atp), by InvivoGen (1 mentions) Z vad fmk, by Enzo Life Sciences (1 mentions) Kineret, by BioVitrum (1 mentions) Baicalein, by Enzo Life Sciences (1 mentions) Mcc950, by Thermo Fisher Scientific (1 mentions) Ly255283, by Cayman Chemical (1 mentions) Recombinant mouse il 1β, by R&D Systems (1 mentions) U75302, by Enzo Life Sciences (1 mentions) Dimethyl sulfoxide (dmso), by Merck Group (1 mentions) Z devd fmk, by Merck Group (1 mentions) Mito tempo, by Santa Cruz Biotechnology (1 mentions) Lipopolysaccharides (lps), by Merck Group (1 mentions) Lps from escherichia coli 0111 b4, by Merck Group (1 mentions) Ca 074me, by Enzo Life Sciences (1 mentions)

10 protocols using ac yvad cmk

1

Investigating HIV-1 gp120 and Methamphetamine

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Full-length HIV-1MN gp120 (gp120, Clade B) was purchased from Immunodiagnostics, Inc. (Woburn, MA). Aliquots of gp120 were kept as 100 nM stock solution at −80 °C. The stock solution was diluted to desired concentrations with artificial cerebrospinal fluid (ACSF) 2–5 min before test. Methamphetamine was purchased from Sigma-Aldrich (St. Louis, MO, Cat # M-8750) with DEA license # RX0374974. Lipopolysaccharide (LPS, from Escherichia coli 0111:B4) was also purchased Sigma-Aldrich. Ac-YVAD-CMK was obtained from Enzo Life Sciences (Farmingdale, NY). All other chemicals, unless otherwise specified, were purchased from Sigma-Aldrich.
Zheng Y., Reiner B., Liu J., Xu L, & Xiong H. (2022). Methamphetamine augments HIV-1 gp120 inhibition of synaptic transmission and plasticity in rat hippocampal slices: Implications for methamphetamine exacerbation of HIV-associated neurocognitive disorders. Neurobiology of disease, 168, 105712.
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2

Anthrax Lethal Toxin Response in BMDMs

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BMDMs were generated by culturing mouse bone marrow cells in L-cell-conditioned IMDM supplemented with 10% FBS, 1% non-essential amino acid and 1% penicillin–streptomycin for 6 days. BMDMs were seeded in 12-well plates, and the next day either left untreated or stimulated with LPS (5 μg ml−1) for 3 h prior to treatment with different concentrations of anthrax PA, and wild-type or proteolytically inactive LF (Quadratech). In some experiments, BMDMs were pretreated with the caspase-1 inhibitor Ac-YVAD-cmk (50 μM; Enzo Life Sciences) for 30 min prior to LeTx incubation. Alternatively, macrophages were primed with LPS for 3 h and then stimulated with 20 μM nigericin for 1 h or 5 mM ATP for 30 min. In other experiments, BMDMs were infected with S. typhimurium at m.o.i. of 10, 5 or 1 for 3 h in a CO2 incubator at 37 °C, the last hour of which in the presence of gentamycin (50 μg ml−1). Alternatively, BMDMs were infected with F. tularensis at m.o.i. 30 for 3 h, the last hour of which in the presence of gentamycin (50 μg ml−1). Finally, cytosolic dsDNA delivery was performed by transfecting cells for 24 h with Lipofectamine-2000 reagent (Life Technologies) according to the manufacturer’s instructions.
Van Opdenbosch N., Gurung P., Vande Walle L., Fossoul A., Kanneganti T.D, & Lamkanfi M. (2014). Activation of the NLRP1b inflammasome independently of ASC-mediated caspase-1 autoproteolysis and speck formation. Nature Communications, 5, 3209.
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3

Modulation of Inflammatory Cytokine Release

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Cells were seeded into 24 well plates at a density of 1x106 viable cells per well. Each well contained RPMI medium supplemented with 10% fetal bovine serum (FBS), 50 IU/mL penicillin, and 50μg/mL streptomycin (Invitrogen, Frederick MD). Cells for inhibition studies were pre-incubated with the following inhibitors for 30 minutes with 50 μM cathepsin B inhibitor; CA-074Me ([L-3-trans-(Propylcarbamoyl)oxirane-2-carbonyl]-L-isoleucyl-L-proline Methyl Ester), 20 μM pan caspase inhibitor; zVAD-FMK (carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone), 20 μM caspase-1 inhibitor; Ac-YVAD-CMK (N-acetyl-L-tyrosyl-L-valyl-N-[(1S)-1-(carboxymethyl)-3-chloro-2-oxo-propyl]-L-alaninamide); (all from Enzo Life Sciences, Inc. Farmingdale, NY). Cells were stimulated with 50 ng/mL LPS for 150 minutes. ATP (2 mM final concentration) (InvivoGen, San Diego, CA) was added (where indicated) at 150 minutes for an additional 30 minutes. Blood cells were collected by centrifugation, and supernatants were collected and used to measure IL-1β. For IL-1β and IL-1α stimulation experiments cells were pre-incubated with 150 μg/ml anakinra (Kineret; Biovitrum, Stockholm, Sweden) and then stimulated with 10, 30, or 100 pg/ml of either IL-1β or IL-1α.
Edwan J.H., Goldbach-Mansky R, & Colbert R.A. (2015). Identification of IL-1β-producing monocytes that are susceptible to pyronecrotic cell death in patients with NOMID. Arthritis & rheumatology (Hoboken, N.J.), 67(12), 3286-3297.
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4

Investigating NLRP3 Inflammasome Inhibition

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Recombinant mouse IL-1β was obtained from R&D Systems (Minneapolis, MN, USA). MCC950 (NLRP3 inhibitor, cat no. inh-mcc) was obtained from Invitrogen (San Diego, CA, USA). Lipopolysaccharide (LPS, Escherichia coli serotype O55:B5), dexamethasone (cat no. D4902), and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, USA). MK886 (5-LOX inhibitor, cat no. 475889) was obtained from Calbiochem (La Jolla, CA, USA). Ac-YVAD-cmk (caspase-1 inhibitor, cat no. ALX-260-028), U75302 (BLT1 antagonist, BML-RA102-0100) and baicalein (12-LOX inhibitor, BML-EI106-0050) were obtained from Enzo Life Sciences (Farmingdale, NY, USA). LY255283 (BLT2 antagonist, cat no. 70715) was purchased from Cayman Chemical (Ann Arbor, MI, USA). Antibodies against NLRP3 and caspase-1 were purchased from AdipoGen Life Sciences (San Diego, CA, USA).
Kwak D.W., Park D, & Kim J.H. (2021). Leukotriene B4 Receptors Are Necessary for the Stimulation of NLRP3 Inflammasome and IL-1β Synthesis in Neutrophil-Dominant Asthmatic Airway Inflammation. Biomedicines, 9(5), 535.
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5

Investigating Pyroptosis Pathways

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To confirm the role of mtROS and caspase-1 in splenic lymphocytes on the NLRP3/caspase-1 pathway in pyroptosis, we use Mito-TEMPO (mtROS inhibitor, 20 mg/kg, Santa Cruz Biotechnology, USA) dissolved in phosphate-buffered saline (PBS) and administered intraperitoneally 1 h before the high-temperature exposure; ac-YVAD-cmk (caspase-1 inhibitor, 6.5 mg/kg, Enzo Biochem, Inc., USA) dissolved in PBS containing 1% DMSO and injected 1 h before heat stress; and Z-DEVD-FMK (6.5 mg/kg, Sigma-Aldrich, USA) dissolved in PBS containing 1% DMSO and injected 1 h before heat stress.
Wang G., Shen T., Li P., Luo Z., Tan Y., He G., Zhang X., Yang J., Liu J., Wang Y., Tang H., Luo X, & Yang X. (2019). The Increase in IL-1β in the Early Stage of Heatstroke Might Be Caused by Splenic Lymphocyte Pyroptosis Induced by mtROS-Mediated Activation of the NLRP3 Inflammasome. Frontiers in Immunology, 10, 2862.
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6

Evaluating NLRP3 Inflammasome Inhibitor in Endotoxemia

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After 2 weeks of adaptation to the new environment, the mice were randomly assigned to control group, YVAD group, LPS group, and YVAD + LPS group based on our established protocol for group randomization (Chang et al., 2014 (link); Wang et al., 2015b ; Wu et al., 2015 (link)). The mice in the control group were injected intraperitoneally with phosphate-buffered saline (PBS) at 10 mL/kg body weight. The mice in the YVAD group were injected intraperitoneally with the NLRP3 inflammasome inhibitor Ac-YVAD-CMK (8 mg/kg body weight, Enzo Life Sciences, Inc., Farmingdale, NY, USA). The mice in the LPS group were injected intraperitoneally with LPS (5 mg/kg body weight, serotype 055:B5, Sigma, St. Louis, MO, USA) (Anderson et al., 2015 ). The mice in the YVAD + LPS group were administered Ac-YVAD-CMK (8 mg/kg body weight) 30 min before LPS (5 mg/kg body weight) injection. Both Ac-YVAD-CMK and LPS were freshly prepared on the treatment day and administered intraperitoneally in a final injection volume of 0.1–0.15 mL. Mice were killed on days 3 and 29 after LPS injection. Results were analyzed in a blinded manner. Based on published studies and our preliminary results, we chose days 3, 28, and 29 after LPS injection to evaluate biochemistry and behavioral parameters (Anderson et al., 2015 ) (Fig. 1).
ZHU W., CAO F.S., FENG J., CHEN H.W., WAN J.R., LU Q, & WANG J. (2016). NLRP3 INFLAMMASOME ACTIVATION CONTRIBUTES TO LONG-TERM BEHAVIORAL ALTERATIONS IN MICE INJECTED WITH LIPOPOLYSACCHARIDE. Neuroscience, 343, 77-84.
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7

Methamphetamine and LPS Stimulation

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(+)-Methamphetamine (D-Meth), (−)-Methamphetamine (L-Meth) and LPS (from Escherichia coli 0111:B4) were purchased from Sigma-Aldrich (St. Louis, MO). Ac-YVAD-CMK, CA-074Me, and Mito-TEMPO were obtained from Enzo Life Sciences (Farmingdale, NY). All chemicals, unless otherwise specified, were from Sigma-Aldrich.
Xu E., Liu J., Liu H., Wang X, & Xiong H. (2018). Inflammasome activation by methamphetamine potentiates lipopolysaccharide stimulation of IL-1β production in microglia. Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology, 13(2), 237-253.
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8

Investigating Innate Immune Responses

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Lipopolysaccharide from E. coli Serotype EH100(Ra) was purchased from Enzo. Lipoteichoic acid from Bacillus subtilis, peptidoglycan from Staphylococcus aureus, R848, and Pam3CSK4 were purchased from Invivogen. Ac-YVAD-CMK (inhibitor of caspase-1) and CRID 3 (inhibitor of NLRP3) were purchased from Enzo and Tocris, respectively. Recombinant mCherry was purchased from BioVision. Pitstop-2 was purchased from Abcam. Wortmannin, methyl-β-cyclodextrin, ATP, nigericin and human fibrinogen were purchased from Sigma.
Valderrama J.A., Riestra A.M., Gao N.J., LaRock C.N., Gupta N., Ali S.R., Hoffman H.M., Ghosh P, & Nizet V. (2017). Group A Streptococcal M Protein Activates the NLRP3 Inflammasome. Nature microbiology, 2(10), 1425-1434.
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9

Pyroptosis Pathway Regulation Assay

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Unless noted, all reagents were from Thermo Fisher Scientific (Waltham, MA, USA). FCA, nigericin, and Ponceau S were from Sigma-Aldrich (St. Louis, MO, USA). PV was from TCI America (Portland, OR, USA). Ac-YVAD-cmk was from Enzo Life Sciences (Farmingdale, NY, USA). Fam-YVAD-fmk (FLICA) was from ImmunoChemistry Technologies (Bloomington, MN, USA). Ultrapure LPS was from Invivogen (San Diego, CA, USA). One endotoxin unit per milliliter Ultrapure LPS is approximately equal to 1 ng/mL from the same manufacturer. GelRed was from Phenix Research Products (Candler, NC, USA). SLO was purified, and hemolytic activity was assessed as previously described (49 (link)). The specific activity of SLO was 1.07 × 106 HU/mg. Murine IL-1β cloned into pFB-Neo retroviral vector (Agilent, Santa Clara, CA, USA) using NotI and EcoRI was obtained from Dr. Chenqun Sun (University of Pittsburgh). Murine Casp1 was cloned into peCFP-N1 (Clontech, Mountain View, CA, USA) using XhoI and NotI, which removed the fluorescent protein. Human Dnase1L3 was cloned into pIRES-DsRed (Clontech) using EcoRI and BamHI sites. The R206C mutation was made in Dnase1L3 via Quikchange mutagenesis (Agilent, Santa Clara, CA, USA). PCR primers were from Integrated DNA Technologies (Coralville, IA, USA) with sequences available upon request.
Shi G., Abbott K.N., Wu W., Salter R.D, & Keyel P.A. (2017). Dnase1L3 Regulates Inflammasome-Dependent Cytokine Secretion. Frontiers in Immunology, 8, 522.
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10

Investigating HIV-1 gp120 and Methamphetamine

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Full-length HIV-1MN gp120 (gp120, Clade B) was purchased from Immunodiagnostics, Inc. (Woburn, MA). Aliquots of gp120 were kept as 100 nM stock solution at −80 °C. The stock solution was diluted to desired concentrations with artificial cerebrospinal fluid (ACSF) 2–5 min before test. Methamphetamine was purchased from Sigma-Aldrich (St. Louis, MO, Cat # M-8750) with DEA license # RX0374974. Lipopolysaccharide (LPS, from Escherichia coli 0111:B4) was also purchased Sigma-Aldrich. Ac-YVAD-CMK was obtained from Enzo Life Sciences (Farmingdale, NY). All other chemicals, unless otherwise specified, were purchased from Sigma-Aldrich.
Zheng Y., Reiner B., Liu J., Xu L, & Xiong H. (2022). Methamphetamine augments HIV-1 gp120 inhibition of synaptic transmission and plasticity in rat hippocampal slices: Implications for methamphetamine exacerbation of HIV-associated neurocognitive disorders. Neurobiology of disease, 168, 105712.
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