B-27, L-glutamic acid, deoxyribonuclease (DNase), fluorescent dye, anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), soybean trypsin inhibitor, fluo-8, and poly-l-lysine (PLL) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Anti-p-p38 MAPK (p-p38), anti-p-JNK, anti-p-p44/42 MAPK (p-ERK1/2), anti-p38 MAPK, anti-JNK, anti-ERK1/2, and anti-rabbit/mouse IgG antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Donkey anti-rabbit/mouse secondary antibodies and Alexa Fluor 488 were obtained from Jackson ImmunoResearch Laboratories, Inc., (PA, USA). Fetal bovine serum, neurobasal medium, RPMI 1640 medium, and trypsin were purchased from Gibco (Gaithersburg, MD, USA). The Gαq inhibitor YM 254,890 was obtained from Wako Pure Chemical Industries (Osaka, Japan). L-741,626, SCH 23390, NMDA, and PP2, LY 23,959 were purchased from Tocris Bioscience (Ellisville, MO, UK). YM 254,896 and PP2 were dissolved in 2% DMSO, L-741,626 was dissolved in 25% DMSO, SCH 23390, NMDA and LY2359549 were dissolved in sterilized saline.
Ym 254890
Manufactured by Fujifilm
Sourced in Japan, United States
The YM-254890 is a laboratory equipment product manufactured by Fujifilm. It serves as a core functional component in various scientific and research applications. The detailed technical specifications and intended uses of this product are not available for inclusion in this response.
Automatically generated - may contain errors
Lab products found in correlation
Rpmi 1640 medium, by Thermo Fisher Scientific (3 mentions)
U73122, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Anti glyceraldehyde 3 phosphate dehydrogenase gapdh, by Merck Group (1 mentions)
Fluo 8, by Merck Group (1 mentions)
Anti p p44 42 mapk p erk1 2, by Cell Signaling Technology (1 mentions)
Rabbit anti mouse igg, by Cell Signaling Technology (1 mentions)
L 741 626, by Bio-Techne (1 mentions)
Sch23390, by Bio-Techne (1 mentions)
Anti p38 mapk, by Cell Signaling Technology (1 mentions)
Anti erk1 2, by Cell Signaling Technology (1 mentions)
Anti jnk, by Cell Signaling Technology (1 mentions)
Neurobasal medium, by Thermo Fisher Scientific (1 mentions)
Soybean trypsin inhibitor, by Merck Group (1 mentions)
L glutamic acid, by Merck Group (1 mentions)
Alexa fluor 488, by Jackson ImmunoResearch (1 mentions)
Butyrate, by Merck Group (1 mentions)
Pertussis toxin ptx, by List Biological Laboratories (1 mentions)
19 protocols using ym 254890
1
Signaling Pathway Analysis in Neuronal Cells
2
Investigating Gαq Inhibition on Explants
3
Butyrate Modulation of CIEC Proliferation
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CIECs were (ATCC Cell Bank, Shanghai, China) prepared with DMEM, including 10% fetal bovine serum (FBS) and 1% antibiotics. Next, different butyrate concentrations (0, 0.1, 0.5, 2 or 5 mM; Sigma-Aldrich, St. Louis, MO, USA) were added into the medium and incubated at 37 °C in 5% CO2 for 24 h. The proliferation level of the CIECs was determined by methyl thiazolyl tetrazolium (MTT) assay. We used the stimulation index (SI) to express the proliferative activity of CIECs. SI = OD570 (stimulated cells)/OD570 (unstimulated cells).
In addition, to explore the intracellular signaling pathway activated by butyrate, we added 10 μM LY294002 (an inhibitor of PI3k, MCE, Weehawken, NJ, USA), 2 μM GSK690693 (an inhibitor of AKT, MCE, Weehawken, NJ, USA), 5 μM CHIR99021 (an inhibitor of GSK-3β, MCE, Weehawken, NJ, USA), 20 μM 4-CMTB (an agonist of GPR43, MCE, Weehawken, NJ, USA), 5 μM AR420626 (an agonist of GPR41, GlpBio, Montclair, NJ, USA), 50 ng/mL PTX (an inhibitor of Gi, List Biological Laboratories, Campbell, CA, USA) and 10 μM Ym254890 (an inhibitor of Gq, Wako Pure Chemical Industries, Ltd. Osaka, Japan), respectively, into CIECs for 0.5 h before the addition of exogenous butyrate (0.5 mM, Sigma-Aldrich, St. Louis, MO, USA). Twenty-four hours later, CIECs were collected for western blot assay. Each assay used a repeat of six wells.
In addition, to explore the intracellular signaling pathway activated by butyrate, we added 10 μM LY294002 (an inhibitor of PI3k, MCE, Weehawken, NJ, USA), 2 μM GSK690693 (an inhibitor of AKT, MCE, Weehawken, NJ, USA), 5 μM CHIR99021 (an inhibitor of GSK-3β, MCE, Weehawken, NJ, USA), 20 μM 4-CMTB (an agonist of GPR43, MCE, Weehawken, NJ, USA), 5 μM AR420626 (an agonist of GPR41, GlpBio, Montclair, NJ, USA), 50 ng/mL PTX (an inhibitor of Gi, List Biological Laboratories, Campbell, CA, USA) and 10 μM Ym254890 (an inhibitor of Gq, Wako Pure Chemical Industries, Ltd. Osaka, Japan), respectively, into CIECs for 0.5 h before the addition of exogenous butyrate (0.5 mM, Sigma-Aldrich, St. Louis, MO, USA). Twenty-four hours later, CIECs were collected for western blot assay. Each assay used a repeat of six wells.
4
Characterization of MRGPRX2 Signaling
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All cell culture reagents were obtained from Invitrogen (Gaithersburg, MD, USA). Amaxa transfection kit (Kit V) was obtained from Lonza (Gaithersburg, MD, USA). Q5 Site-Directed Mutagenesis Kit was from New England BioLabs (Ipswich, MA). Substance P (SP) was from AnaSpec (Fremont, CA, USA). Pertussis toxin (PTx) was from List Biological Laboratories (Campbell, CA, USA). YM-254890 was from Wako Chemicals (Richmond, VA, USA) and p-nitrophenyl-N-acetyl-β-D-glucosamine (PNAG) was from Sigma-Aldrich (St. Louis, MO, USA). Fura-2 acetoxymethyl ester was from Abcam (Cambridge, MA, USA). PE-conjugated anti-MRGPRX2 antibody was from BioLegend (San Diego, CA, USA). MRGPRX2 plasmid encoding hemagglutinin (HA)-tagged human MRGPRX2 in pReceiver-MO6 vector was obtained from GeneCopoeia (Rockville, MD, USA).
5
AngII-induced Ch25h expression in VSMCs
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Before the experiments, the VSMCs were serum deprived overnight using DMEM supplemented with 0.1% bovine serum albumin (BSA; Sigma-Aldrich). VSMCs were pretreated with either dimethyl sulfoxide (DMSO) vehicle or various inhibitors separately: 10 µM candesartan, 1 µM JNK-IN-8 (IN-8), 50 µM SB202190, 20 µM PD98059 (Sigma-Aldrich), 1 µM YM-254890 (Wako Chemicals), and 5 µM DPI (Sigma-Aldrich). Pretreatment lasted for 30 min. Following pretreatment, VSMCs were stimulated with 100 nM AngII or vehicle for 1 h. In the other sets of experiments, the VSMCs were stimulated with either vehicle or 3 µM TRV120023 (TRV3; Proteogenix) for 1 h. To assess time dependency of Ch25h mRNA expression, VSMCs were stimulated with 100 nM AngII for 1, 2, 3, 4, 5, and 6 h or not stimulated.
For the detection of 25-HC, VSMCs were stimulated with 1 µM AngII for 2, 4, 8, 16, and 24 h or not stimulated. The experiments were performed in duplicates. After the hormone stimulation, the supernatants were collected and subjected to LC-MS/MS measurement.
For the detection of 25-HC, VSMCs were stimulated with 1 µM AngII for 2, 4, 8, 16, and 24 h or not stimulated. The experiments were performed in duplicates. After the hormone stimulation, the supernatants were collected and subjected to LC-MS/MS measurement.
6
Influenza Hemagglutinin Epitope Detection
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Cell culture materials were purchased from Invitrogen, FR900359 (previous commercial name UBO-QIC) was isolated and purified as described elsewhere (24 (link)), YM-254890 was from Wako Chemicals GmbH. Primary antibodies to detect the human influenza hemagglutinin (HA) epitope tag (YPYDVPDYA) and α-tubulin were from Roche Applied Science and LifeSpan BioSciences, respectively. The horseradish peroxidase–conjugated secondary antibodies, goat anti-mouse IgG, and goat anti-rabbit IgG were from Sigma-Aldrich and Antibodies-online GmbH, respectively. All other reagents were purchased from Sigma-Aldrich if not stated otherwise.
7
Measuring cAMP and Ca2+ in Rhodopsin-HEK293S Cells
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The intracellular cAMP and Ca2+ levels in rhodopsin-expressing HEK293S cells (human embryonic kidney 293 S cells, provided by Dr. Jeremy Nathans of Johns Hopkins University) were measured using the GloSensor cAMP assay and the aequorin assay, respectively, as described previously (Bailes and Lucas, 2013 (link)). HEK293S cells have been confirmed to be free from mycoplasma contamination. The identity of HEK293S cells was confirmed by similarity to HEK293 and HEK293T cells through STR profiling, and by morphological observation of the cells. The pGloSensor-20F cAMP plasmid (Promega) was used for the GloSensor cAMP assay. The wild type aequorin obtained by introducing two reverse mutations into the plasmid [pcDNA3.1+/mit-2mutAEQ] (Addgene #45539) (de la Fuente et al., 2012 (link)) was used for the aequorin assay. The rhodopsin expression plasmids were constructed based on pCS2+ (see the Zebrafish section) and used for transfection. For Gαq inhibition, YM-254890 (FUJIFILM Wako Pure Chemical Corp., 257–00631, Osaka, Japan) was added (1 μM) 5 min before the measurement. Green (500 nm) and violet (410 nm) LED lights were applied for 5 s in the GloSensor cAMP assay and for 1 s in the aequorin assay as light stimuli. Dual Head LED Light 505 nm (GB Life Science) and SPL-25-CC (REVOX, Inc) were used for green and violet LED light stimulation, respectively.
8
AngII Receptor Binding Assay
9
Culturing and Transfecting Cell Lines
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A431 cells [Japanese Collection of Research Bioresources Cell Bank (JCRB)], EpH4 cells (a gift from E. Reichmann, University Children’s Hospital Zurich, Switzerland), L cells, and HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS). MCF7 cells (RIKEN BRC) were cultured in Eagle’s Minimum Essential Medium (Wako) supplemented with 10% FCS and 1% nonessential amino acids. HT29 cells (American Type Culture Collection) were cultured in McCoy’s 5A (Gibco) supplemented with 10% FCS. Cell cultures were incubated at 37°C in a humidified 5% CO2 atmosphere. cDNA transfection was performed using the Lipofectamine LTX Reagent (Invitrogen) according to the manufacturer’s instructions. The following G alpha agonists and antagonists were used: cholera toxin A subunit (036-20601, Wako), P. multocida (01-507, BioAcademia), pertussis toxin (168-22471, Fujifilm/Wako), NF-449 (1391/10, R&D Systems), and YM-254890 (257-000631, Fujifilm/Wako).
10
Mast Cell Activation Assay Reagents
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Lithocholic acid (LCA), deoxycholic acid (DCA), tauroLithocholic acid (TLCA), compound 48/80, IL-3, U-73122, SKF-96365, and 4-Nitrophenyl N-acetyl-β-D-glucosaminide (pNAG) were purchased from Sigma-Aldrich (St. Louis, MO, USA). YM-254890 was purchased from FUJIFILM Wako (Osaka, Japan). QWF (Boc-Gln-D-Trp(Formyl)-Phe benzyl ester trifluoroacetate) was obtained from Tocris Bioscience (Bristol, UK). Murine stem cell factor (SCF) was purchased from Peprotech (Cranbury, NJ, USA).
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