Fetal ASM were incubated at humidified 95% air/5% CO2 in DMEM/F12 phenol red free medium, 10% FBS, 1% antibiotic-antimycotic (Life Technologies, Carlsbad, CA) and grown to 80% confluence prior to experiments. Cells were serum starved in 0.5% FBS medium for 24 h prior to all studies. The next day, fresh 0.5% FBS media was added to plates and cells were treated with media only, 100 µM dibutyl-cAMP (cell-permeant cAMP analog; EMD Millipore, Billerica, MA), 10µM Roflumilast (PDE4 inhibitor; Cayman Chemical, Ann Arbor, MI), 0.5 µM KT5720 (PKA inhibitor; EMD Millipore), or 10 µM 8-CPT-2-O-Me-cAMP-AM (Epac-selective cAMP analog; Tocris Bioscience, U.K.) for 30 min and then exposed to room air (21% O2) or hyperoxia (50 % O2) for 8, 12, 24, or 48 h. Oxygen levels were monitored using a MiniOx 3000 oxygen monitor (MSA Medical Products, Pittsburgh, PA). Media or cell lysates were collected and stored at −80°C until analyses.
Kt5720
Manufactured by Merck Group
Sourced in United States, Germany, Japan
KT5720 is a laboratory product manufactured by the Merck Group. It is a compound used in scientific research and experimentation. The core function of KT5720 is to serve as a tool for researchers and scientists in their investigations and experiments.
Automatically generated - may contain errors
Lab products found in correlation
Forskolin, by Merck Group (12 mentions)
Ly294002, by Merck Group (6 mentions)
Kt5823, by Merck Group (5 mentions)
8 br camp, by Merck Group (4 mentions)
Cycloheximide (chx), by Merck Group (4 mentions)
Isoproterenol, by Merck Group (3 mentions)
Sq22536, by Merck Group (3 mentions)
U0126, by Merck Group (3 mentions)
Pd98059, by Merck Group (3 mentions)
3 isobutyl 1 methylxanthine, by Merck Group (3 mentions)
Arbutin, by Merck Group (3 mentions)
U73122, by Merck Group (3 mentions)
3 isobutyl 1 methylxanthine ibmx, by Merck Group (3 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Rolipram, by Merck Group (2 mentions)
Esi 09, by Merck Group (2 mentions)
Capsaicin, by Merck Group (2 mentions)
Thapsigargin, by Merck Group (2 mentions)
Ns 398, by Merck Group (2 mentions)
60 protocols using kt5720
1
Fetal ASM Hyperoxia Response Assay
2
Isoproterenol-Induced Hypertrophy in H9C2 Cardiomyoblasts
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The rat cardiomyoblast cell line (H9C2) was purchased from ATCC®. Cells in culture were treated for 1–24 h with isoproterenol (1 μmol/L, Sigma-Aldrich) or vehicle. Selective β1- or β2-antagonist, atenolol, and ICI-118551 (both at 1 μmol/L, Sigma-Aldrich) were tested. Expression of VEGF was measured by real-time RT-PCR with results normalized by the reference gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH). To test signaling mechanisms for isoproterenol regulated VEGF expression, we tested effects of the PKA inhibitor KT5720 (0.3 μmol/L), Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitory peptide myristoylated-AIP (10 μmol/L; Merck-Millipore, Darmstadt, Germany) or inhibitor KN93 (10 μmol/L; Calbiochem, Merck-Millipore). To determine onset of isoproterenol-induced hypertrophic growth, cell size was determined using Image J (NIH, USA) and expression at mRNA level of atrial natriuretic peptide (ANP) was measured. Expression at protein level of p53 was determined by immunoblotting using antibody from Santa Cruz Biotechnology.
3
Amyloid-β Aggregation Assay Protocol
4
Melanoma Cell Line Inhibition Assay
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The melanoma cell line Mel Ei (150,000 cells) were cultured in six-well plates and were treated for 24 h with different inhibitors (SB431542 InvivoGen 2 μM; S3I-201 Sigma-Aldrich 20 mM; Dorsomorphin Tebi-bio 2 mM; LY-294002 Sigma-Aldrich 20 mM; Wortmannin Sigma-Aldrich 10 mM; UO126 Calbiochem 10 mM; Vemurafenib Active Biochem 100 μM; KT5720 Merck Millipore 100 nM; Bisindolylmaleimide II Santa Cruz 10 μM; Gö 6983 Santa Cruz 10 μM; Wnt Agonist Calbiochem 10 μM). For the stability assay the cells were treated with α-Amanitin (AppliChem, 5 mM). After isolation of total RNA, quantitative RT-PCR was performed to detect relative gene expression. Normalization was based on mRNA input in this experiment.
5
Extracellular Vesicles Modulate Mitochondrial Dynamics
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After 48 h of fibroblast culture, the culture medium from a 25 mL culture flask was collected and extracellular vesicles were purified according to the method described in [15 (link)]. HL-1 cells were transfected using CellLight™ Mitochondria-GFP, BacMam 2.0 (ThermoFisher Scientific, Waltham, MA, USA) and incubated with the vesicles for 24 h. Alternatively, the cells were preincubated with 10 µM forskolin (Merck, Darmstadt, Germany) before application of EVs or treated with 2 µM PKA inhibitor (KT5720, Merck, Darmstadt, Germany) for 24 h. The velocity of at least 144 mitochondria was measured for each treatment. The experiment was performed in duplicate.
To test the effect of PKA inhibitor on the ability of nocodazole to destabilize microtubules, HL-1 cells were pretreated with a 2 µM PKA inhibitor for 15 min and then incubated with 0.1 µM nocodazole (Merck, Darmstadt, Germany) for 1 h. Cells were fixed, and microtubules were stained as described in the Immunofluorescence subsection.
To test the effect of PKA inhibitor on the ability of nocodazole to destabilize microtubules, HL-1 cells were pretreated with a 2 µM PKA inhibitor for 15 min and then incubated with 0.1 µM nocodazole (Merck, Darmstadt, Germany) for 1 h. Cells were fixed, and microtubules were stained as described in the Immunofluorescence subsection.
6
Modulation of TRPV1-mediated Knee Pain
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To examine TRPV1 function in rat knee joints, Capsaicin-induced pain-related behavior was evaluated on day 14 after MIA or saline injection. Rats were randomly allocated by body weight and grip strength. Behavioral tests were conducted during the light phase. Capsaicin (SigmaeAldrich) was dissolved into 50 mL 10% ethanol in saline and injected into the articular cavity of the right leg knee joint. Response behavior was measured by duration of flinching and licking behavior for 10 min in a plexiglas cylinder (25 cm diameter, 30 cm high) after Capsaicin injection by a blinded investigator. Bisindolylmaleimide I (PKC inhibitor, EMD Millipore, Billerica, MA, USA) and KT5720 (PKA inhibitor, EMD Millipore) were dissolved in 50 mL of 10% DMSO in saline and administered before Capsaicin injection. Phorbol 12-myristate 13-acetate (PMA) (50 mL, Invivogen, San Diego, CA, USA) was administered before Capsaicin injection. Drugs were administered into the knee joint cavity to examine the effect of those in knee joint. Rats were tested in order of allocated groups.
7
Modulation of Cellular Signaling Pathways
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Zinterol, AACOCF3, GW5074, edelfosine (ET-18-OCH3), chelerythrine, 2-aminoethyl diphenyl borate (2-APB), KT5720, methyl-β-cyclodextrin, ryanodine, thapsigargin (Sigma Chemical).
8
Investigating Neuroinflammation Pathways
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PGI2, PGE2, Aβ1–42 and the inhibitors NS398, U0126, and KT5720 were obtained from Sigma-Aldrich Corp (St. Louis, MO, USA). Antibodies against β-actin, ERK1/2, p-ERK1/2 (Thr 202/Tyr 204), NF-κB, p-NF-κB (Ser 536), p-NF-κB (Ser 276), IκB, IFNγ, BACE-1, PS1, PS2, GFAP and human Aβ were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). sAPPα and sAPPβ antibodies were obtained from IBL International Corp. (Toronto, ON, Canada). The human IFNγ and IFNγ enzyme immunoassay kits were obtained from Raybiotech, Inc. (Norcross, GA, USA). Human or mouse Aβ1–42 ELISA kits were obtained from Invitrogen (Carlsbad, CA, USA). ERK1/2, p65 and scramble siRNA were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). The chromatin immunoprecipitation (ChIP) EZ-ChIP kit was purchased from Upstate Biotechnology. All reagents for the qRT-PCR and SDS-PAGE experiments were purchased from Bio-Rad Laboratories. All other reagents were from Invitrogen (Carlsbad, CA, USA) unless otherwise specified.
9
Cellular Assay Reagents and Conditions
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The following reagents were used in the present study: carboplatin, poly-D-lysine, U73122, KT5720, forskolin, and Hank’s balanced salt solution (Sigma-Aldrich, St Louis, MO, USA); fetal bovine serum (Gibco, Carlsbad, CA, USA); penicillin/streptomycin and AITC (Nacalai Tesque, Kyoto, Japan); blasticidin S and zeocin (Invitrogen, Carlsbad, CA, USA); acetone, 0.25% trypsin-EDTA, Dulbecco’s modified Eagle’s medium and tetracycline (Fujifilm Wako Pure Chemical, Osaka, Japan); fura-2 acetoxymethyl ester (Dojindo Laboratories, Kumamoto, Japan); AKAP I (Tocris Bioscience, Bristol, UK). All other reagents were of the highest purity available from commercial sources.
10
Serotonin Signaling Pathway Modulation
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The following drugs and substances were used (respective suppliers in parentheses): Serotonin (5-hydroxytryptamine, 5-HT), selective p38MAPK inhibitor SB 220025, selective PKA inhibitor KT 5720 and selective inhibitor of ERK pathway PD98059, (Sigma–Aldrich; St. Louis, MO, USA). Pam3CSK4 and Pam2CSK4, specific TLR2/1 and TLR2/6 agonists respectively (InvivoGen; San Diego, CA, USA). [3H]-5-HT (specific activity 25–30 Ci/mM) (Perkin-Elmer; Boston, MA, USA). Primary antibodies used were: goat polyclonal antibody anti-human and anti-mouse SERT (ab130130) and rabbit monoclonal anti-human TLR2 (ab108998) (Abcam, Cambridge, UK); mouse monoclonal anti-human p38 (sc-7972), rabbit polyclonal antibody anti-human pp38 (sc-7975-R) and secondary antibodies coupled to horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA, USA). All generic reagents were purchased from Sigma–Aldrich and Roche Applied Sciences (Sant Cugat del Vallés, Barcelona, Spain).
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