R59949

Manufactured by Merck Group

Sourced in Germany, China

R59949 is a laboratory instrument manufactured by Merck Group. It is designed to perform specific laboratory functions. The core function of this product is to assist in scientific research and analysis, but the detailed specifications and intended use are not available for this response.

Automatically generated - may contain errors

Lab products found in correlation

Ly341495, by Bio-Techne (2 mentions) Ly357385, by Bio-Techne (2 mentions) Gefitinib, by Selleck Chemicals (2 mentions) Dimethyl malonate, by Merck Group (2 mentions) Osimertinib, by Selleck Chemicals (2 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions) Ritanserin, by Merck Group (2 mentions) R59022, by Merck Group (2 mentions) U73122, by Merck Group (2 mentions) Fetal calf serum (fcs), by Lonza (2 mentions) Antibiotics antimycotics, by Merck Group (2 mentions) R cpp, by Bio-Techne (1 mentions) Mitotracker red cmxros, by Thermo Fisher Scientific (1 mentions) Cycloheximide (chx), by Merck Group (1 mentions) Rapamycin, by Merck Group (1 mentions) Nocodazole, by Merck Group (1 mentions) Brefeldin a, by Illumina (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Hepg2, by American Type Culture Collection (1 mentions) Hepg2, by Merck Group (1 mentions)

13 protocols using r59949

Sequential Drug Wash-in Protocol

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Sequential drug wash-ins were performed using a computer-controlled solenoid manifold system (ValveLink 8.2, Automate Scientific, Inc., Berkeley, CA) with a flow rate of 1 to 2 mL/min, where indicated, with mGluR1 antagonist (100 µM LY357385, Tocris Bio-Techne, Minneapolis, MN), AMPA receptor blocker (5 µM NBQX), NMDA receptor blocker (2 µM R-CPP), mGluR2/3 antagonist (1 µM LY341495, Tocris Bio-Techne, Minneapolis, MN) and DGK inhibitor II (100 µM R59949, MilliporeSigma, St. Louis, MO)

Guo C., Huson V., Macosko E.Z, & Regehr W.G. (2021). Graded heterogeneity of metabotropic signaling underlies a continuum of cell-intrinsic temporal responses in unipolar brush cells. Nature Communications, 12, 5491.

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Sequential Drug Delivery Manifold

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Sequential drug wash-ins were performed using a computer-controlled solenoid manifold system (ValveLink 8.2, Automate Scientific, Inc., Berkeley, CA) with a flow rate of 1 to 2mL/min, where indicated, with mGluR1 antagonist (100µM LY357385, Tocris Bio-Techne, Minneapolis, MN), AMPA receptor blocker (5µM NBQX), NMDA receptor blocker (2 µM R-CPP), mGluR2/3 antagonist (1µM LY341495, Tocris Bio-Techne, Minneapolis, MN) and DGK inhibitor II (100 µM R59949, MilliporeSigma, St. Louis, MO)

Guo C., Huson V., Macosko E.Z., & Regehr W.G. (2020). Graded heterogeneity of metabotropic signaling underlies a continuum of cell-intrinsic temporal responses.

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Multimodal Mitochondrial Labeling

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MitoTracker Red CMXRos and Tf-Alexa Fluor 594 was from Thermo Fisher Scientific. PAO and nocodazole were from Merck. Rapamycin, cycloheximide, and R59949 were from Sigma-Aldrich. Brefeldin A was from Epicenter Technologies.

Mahajan D., Tie H.C., Chen B, & Lu L. (2019). Dopey1-Mon2 complex binds to dual-lipids and recruits kinesin-1 for membrane trafficking. Nature Communications, 10, 3218.

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Pharmacological Inhibition of EGFR Signaling

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Osimertinib (S7297) and gefitinib (S1025) were purchased from Selleck Chemicals. DMOG ^{65 (link)} (Cat #400091) was from Calbiochem. R59949 ^{66 (link)} (Cat #D5794) and dimethyl malonate ^{67 (link),68 (link)} (DMM, Cat #136441) were purchased from Sigma-Aldrich.

Zhang W.C., Wells J.M., Chow K.H., Huang H., Yuan M., Saxena T., Melnick M.A., Politi K., Asara J.M., Costa D.B., Bult C.J, & Slack F.J. (2019). miR-147b-mediated TCA cycle dysfunction and pseudohypoxia initiate drug tolerance to EGFR inhibitors in lung adenocarcinoma. Nature metabolism, 1(4), 460-474.

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HepG2 Cell Line Treatment with DGKθ Modulators

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The human liver cancer cell line HepG2 was purchased from American Type Culture Collection (Manassas, VA, USA) and cultured in high-glucose Dulbecco's modified Eagle medium (DMEM; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C, 5% CO₂ (v/v). A total of ~1×10⁵ HepG2 cells were treated with DGKθ inhibitor R59949 at 10 µM, or DGKθ agonist GW4064 (both from Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) at 1 µM for 24 h at 37°C.

Zhang J., Zhao J., Zheng X., Cai K., Mao Q, & Xia H. (2017). Establishment of a novel hepatic steatosis cell model by Cas9/sgRNA-mediated DGKθ gene knockout. Molecular Medicine Reports, 17(2), 2169-2176.

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Pharmacological Inhibition of DGK In Vivo

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For pharmacological inhibition of DGK activity in vivo, mice were intraperitoneally injected once daily with either DMSO or the DGK inhibitor R59949 (10 mg/kg; Sigma-Aldrich) in 50% polyethylene glycol (PEG) 400 (Sigma-Aldrich) solution beginning immediately after the second intraperitoneal OVA/Alum immunization and ending before the last intranasal OVA challenge (late sensitization and challenge phases) or beginning immediately before the first intranasal OVA challenge and ending before the last intranasal OVA challenge (challenge phase only).

Singh B.K., Lu W., Schmidt Paustian A.M., Ge M.Q., Koziol-White C.J., Flayer C.H., Killingbeck S.S., Wang N., Dong X., Riese M.J., Deshpande D.A., Panettieri RA J.r., Haczku A, & Kambayashi T. (2019). Diacylglycerol kinase ζ promotes allergic airway inflammation and airway hyperresponsiveness through distinct mechanisms. Science signaling, 12(597), eaax3332.

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Liposome-based Serotonin Receptor Assay

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[γ³²P]-ATP was from Perkin Elmer (Boston, MA). The diacylglycerol (DAG) species used in this study are as follows: 1,2-dioleoyl-sn-glycerol (dioleoyl; 18:1, 18:1), 1,2-octanoyl-sn-glycerol (dioctanoyl; 8:0, 8:0) and 1-stearoyl-2-arachidonoyl-sn-glycerol (stearoyl arachidonoyl; 18:0 20:4). These DAG species as well as 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine] (PS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (PC), and all materials for the preparation of liposomes were also from Avanti Polar Lipids (Alabaster, AL). M2 FLAG beads, FLAG antibody, rabbit and mouse alkaline-conjugated secondary antibodies, R59949, R59022, and ritanserin were from Sigma-Aldrich (St. Louis, MO). Ketanserin, bisindolylmaleimide II (bis), PMA, and TCB-2 were from Tocris Bioscience (Avonmouth, Bristol, UK). All other commonly used reagents were from Sigma-Aldrich, unless otherwise indicated. All cell lines were obtained from ATCC (Rockville, MD).

Boroda S., Niccum M., Raje V., Purow B.W, & Harris T.E. (2016). Dual activities of ritanserin and R59022 as DGKα inhibitors and serotonin receptor antagonists. Biochemical pharmacology, 123, 29-39.

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Calcium Signaling Pathway Modulation

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Fura-2/AM was obtained from Invitrogen (Life Technologies; cat# F1221). BTP-2 (cat# 203890) and myo-inositol 1,4,5-trisphosphate hexakis (butyryloxymethyl) (InsP₃BM) (cat# 3-1-145) were obtained from Calbiochem (Merk Millipore). All other chemicals were of analytical grade and were obtained from Sigma-Aldrich: cyclopiazonic acid (CPA) (cat# C1530), ATP (cat# A2383), caffeine (cat# C0750), R59949 (cat# D5794), ritanserin (cat# R103), phorbol 12-myristate 13-acetate (PMA) (cat# 79346), Gö-6983 (cat# G1918), U73122 (Cat# U6756, 2-Aminoethyl diphenylborinate (2-APB) (cat# D9754) and ionomycin (cat# I0634).

Faris P., Rumolo A., Tapella L., Tanzi M., Metallo A., Conca F., Negri S., Lefkimmiatis K., Pedrazzoli P., Lim D., Montagna D, & Moccia F. (2022). Store-Operated Ca2+ Entry Is Up-Regulated in Tumour-Infiltrating Lymphocytes from Metastatic Colorectal Cancer Patients. Cancers, 14(14), 3312.

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Signaling Pathway Protein Analysis

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Antibodies against PLCγ1, p-PLCγ1 (Tyr783), PKCδ, CaMK IIβ, Akt, p-Akt (S473), ERK, p-ERK (Thr202/Tyr204), mTOR, p-mTOR (Ser2481), p-S6 (Ser235/236), and NF-κB were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA). Cleaved-PARP, Matrix metalloproteinase-9 (MMP9), and GAPDH were purchased from Abcam (Cambridge, MA, USA). Inhibitors (U73122, KN93, and R59949) were obtained from Sigma-Aldrich in China (Shanghai, China). Other reagents were of the highest grade commercially available.

Dai L., Zhuang L., Zhang B., Wang F., Chen X., Xia C, & Zhang B. (2015). DAG/PKCδ and IP3/Ca2+/CaMK IIβ Operate in Parallel to Each Other in PLCγ1-Driven Cell Proliferation and Migration of Human Gastric Adenocarcinoma Cells, through Akt/mTOR/S6 Pathway. International Journal of Molecular Sciences, 16(12), 28510-28522.

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Isolation and Activation of PBMCs for Apoptosis Assays

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Peripheral blood mononuclear cells (PBMCs) were isolated from normal controls or XLP-1 patients by Ficoll-Paque PLUS (GE Healthcare) density gradient centrifugation, washed, and resuspended at 2×10⁶ cell/ml in complete media (cRPMI): RPMI-GlutaMAX (Life Technologies) containing 10% heat inactivated FCS (Lonza), 2 mM glutamine, and 100 U/ml of penicillin and streptomycin (Life Technologies). T cells were activated with 1 μg/ml anti-CD3 (clone UCHT1) and anti-CD28 (clone CD28.2) antibodies. After 3 days, activated T cells were washed and cultured in cRPMI plus 100 IU/ml rhIL-2 (Peprotech) at 1.2×10⁶ cells/mL for ≥7 days before apoptosis assays were conducted (media changed every 2–3 days).
Jurkat A3 cells were from ATCC, and 293FT from Life Technologies. Cells were cultured in RPMI or DMEM (Life Technologies) with 10% FCS and antibiotics/antimycotics (Sigma-Aldrich).
DGK inhibitors R59949 and R59022 (Sigma-Aldrich) were dissolved in DMSO; all reagents and antibodies used are listed in Table S1.

Ruffo E., Malacarne V., Larsen S.E., Das R., Patrussi L., Wülfing C., Biskup C., Kapnick S.M., Verbist K., Tedrick P., Schwartzberg P.L., Baldari C.T., Rubio I., Nichols K.E., Snow A.L., Baldanzi G, & Graziani A. (2016). Inhibition of diacylglycerol kinase alpha restores restimulation-induced cell death and reduces immunopathology in XLP-1. Science translational medicine, 8(321), 321ra7.

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