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Psb603

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The PSB603 is a laboratory instrument designed for protein purification. It utilizes affinity chromatography to isolate and concentrate specific proteins from complex biological samples.

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Lab products found in correlation

Adenosine, by Merck Group (6 mentions) Zm241385, by Bio-Techne (5 mentions) Cgs15943, by Bio-Techne (5 mentions) Sch58261, by Bio-Techne (4 mentions) Bay 60 6583, by Bio-Techne (4 mentions) Forskolin, by Merck Group (3 mentions) Mrs1220, by Bio-Techne (3 mentions) U73122, by Bio-Techne (2 mentions) Alprenolol, by Merck Group (2 mentions) Isoprenaline, by Bio-Techne (2 mentions) Propranolol, by Bio-Techne (2 mentions) Angiotensin 2, by Merck Group (2 mentions) Alprenolol tamra, by Promega (2 mentions) Tamra angii, by AnaSpec (2 mentions) Dpcpx, by Bio-Techne (2 mentions) Ici 118 551, by Bio-Techne (2 mentions) Cgp 12177, by Bio-Techne (2 mentions) Rp camps, by Bio-Techne (2 mentions) Dpcpx, by Merck Group (2 mentions) Go6983, by Bio-Techne (2 mentions)

23 protocols using psb603

1

Immortalized Murine Mammary Fibroblast Isolation and Characterization

Cited 2 times
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Immortalized mouse mammary fibroblasts were generated in Dr. Harold Moses laboratory (Vanderbilt University), have been used in previous publications [46 (link)–48 (link)], and gifted to us. Primary mouse mammary fibroblasts were isolated from mouse mammary gland by FACS sorting as described below. Fibroblasts were grown in T-75 flasks (Fisher Scientific, USA) in DMEM medium (Gibco, USA) supplemented with 10% FBS, Antibiotic-Antimycotic (Sigma-Aldrich). For protein assessment, fibroblasts were grown in 10cm cell culture dishes (Corning, USA). When cells achieved 90–95% confluency, they were treated with TGFβ, 1ng/ml (R&D System, USA) and 0.01, 0.1, 1, 10, 100 uM NECA (5′-N-Ethylcarboxamido adenosine, Sigma-Aldrich, USA). Following inhibitors were used: for inhibition of A2a adenosine receptors, 1 uM SCH58261(Tocris, USA); for A2b receptors– 1 uM PSB603 (Tocris, USA); for PLC inhibition– 1 uM U73122 (Tocris, USA); for PKA inhibition—1 uM H89 (Tocris, USA); and for activation of adenylate cyclase– 10 uM forskolin (Sigma-Aldrich, USA). Human fibroblast cell lines IMR-90 (CCL-186), WS-1 (CRL-1502), WPMY-1 (CRL-2584), hTERT SMC PM151T (“hTERT” CRL-3291) and BJ (CRL-2522) were purchased from ATCC and cultured according to the manufacturer’s protocol.
Vasiukov G., Menshikh A., Owens P., Novitskaya T., Hurley P., Blackwell T., Feoktistov I, & Novitskiy S.V. (2021). Adenosine/TGFβ axis in regulation of mammary fibroblast functions. PLoS ONE, 16(6), e0252424.
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2

Synthesis and Characterization of Fluorescent Probes

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CA200645, propranolol-BY630 (propranolol-βalanine-βalanine-X-BODIPY-630/650) and propranolol-BYFL (propranolol-βalanine-βalanine-X-BODIPY-FL) were from CellAura. AV039 and ABEA-X-BY630 (ABEA-X-BODIPY-630/650) were synthesised by the University of Nottingham as described by Vernall et al.12 (link) and Middleton et al.14 (link). Alprenolol-TAMRA was synthesised by Promega. TAMRA-AngII was from AnaSpec. Alprenolol and angiotensin II were from Sigma. Candesartan and olmesartan were from Zhou Fang Pharm Chemical. DPCPX, SCH 58261, MRS 1220, CGS 15943, ZM 241385, XAC, PSB 603, isoprenaline, propranolol, ICI 118551 and CGP 12177 were from Tocris.
Stoddart L.A., Johnstone E.K., Wheal A.J., Goulding J., Robers M.B., Machleidt T., Wood K.V., Hill S.J, & Pfleger K.D. (2015). Application of BRET to monitor ligand binding to GPCRs. Nature methods, 12(7), 661-663.
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3

Pharmacological Inhibition of Adenosine Signaling

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Dipyridamole (cat#D9766, Sigma-Aldrich, Merk Life Science, Milan, Italy); 2-(2-Furanyl)-7-(2-phenylethyl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine, SCH58261 (cat#2270, Tocris, Bio-Techne, Milan, Italy); 8-Cyclopentyl-1,3-dipropylxanthine, DPCPX (cat#0439, Tocris, Bio-Techne, Milan, Italy); 8-[4-[4-(4-Chlorophenzyl)piperazide-1-sulfonyl)phenyl]]-1-propylxanthine, PSB603 (cat#3198, Tocris, Bio-Techne, Milan, Italy); 1,4-Dihydro-2-methyl-6-phenyl-4-(phenylethynyl)-3,5-pyridinedicarboxylic acid 3-ethyl-5-[(3-nitrophenyl)methyl] ester, MRS1334 (cat#1385, Tocris, Bio-Techne, Milan, Italy); KT5720 (cat#K3761, Sigma, Merk Life Science, Milan, Italy); PD98059 (car#513001, Calbiochem, Merk Life Science, Milan, Italy); 9-(Tetrahydro-2-furanyl)-9H-purin-6-amine, SQ22536 (cat#1453, Tocris, Bio-Techne, Milan, Italy); (R)-Adenosine, cyclic 3′,5′-(hydrogenphosphorothioate) triethylammonium, Rp-cAMPs (cat#1337, Tocris, Bio-Techne, Milan, Italy).
Pepponi R., De Simone R., De Nuccio C., Visentin S., Matteucci A., Bernardo A., Popoli P, & Ferrante A. (2022). Repurposing Dipyridamole in Niemann Pick Type C Disease: A Proof of Concept Study. International Journal of Molecular Sciences, 23(7), 3456.
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4

Adenosine Receptor Ligands and Analogs

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The adenosine A1 receptor agonist N6-cyclopentyladenosine (N6-CPA, purity > 98%) was obtained from Abcam (Shanghai, China) and dissolved in DMSO at 50 mM. The adenosine A1 receptor antagonist rolofylline (KW-3902, purity > 99%) was obtained from Tocris Bioscience (Bristol, United Kingdom) and dissolved in DMSO at 10 mM. The adenosine A2a receptor agonist CGS 21680 (purity > 98%) was obtained from Abcam and dissolved in DMSO at 10 mM. The adenosine A2a receptor antagonist SCH 58261 (purity > 99%) was obtained from Abcam and dissolved in DMSO at 10 mM. The adenosine A2b receptor agonist BAY 60-6583 (purity > 98%) was obtained from Tocris Bioscience and dissolved in DMSO at 20 mM. The adenosine A2b receptor antagonist PSB603 (purity > 98%) was obtained from Tocris Bioscience and dissolved in DMSO at 10 mM. The adenosine A3 receptor agonist piclidenoson (IB-MECA, purity > 97%) was obtained from Abcam and dissolved in DMSO at 10 mM. The adenosine A3 receptor antagonist MRE 3008F20 (purity > 98%) was obtained from Tocris Bioscience and dissolved in DMSO at 50 mM. The adenosine analog 5′-N-ethylcarboxamidoadenosine (NECA, purity > 99%) was obtained from Tocris Bioscience and dissolved in DMSO at 50 mM. All solutions were stored at –20°C and used within 1 year.
Tang J., Zou Y., Li L., Lu F., Xu H., Ren P., Bai F., Niedermann G, & Zhu X. (2021). BAY 60-6583 Enhances the Antitumor Function of Chimeric Antigen Receptor-Modified T Cells Independent of the Adenosine A2b Receptor. Frontiers in Pharmacology, 12, 619800.
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5

Adenosine Receptor Pharmacology Explored

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APCP (a known 5ʹ-nucleotidase inhibitor), DPCPX (a selective A1 AR antagonist), ZM 241385 (a potent selective A2A AR antagonist), CCPA (a selective A1 AR agonist), AMP, and adenosine were purchased from Sigma-Aldrich. Rp-cAMPS (a competitive antagonist of cAMP-induced PKA activation) and PSB 603 (a potent selective A2B AR antagonist) were purchased from TOCRIS. NECA (a nonselective AR agonist) was purchased from MedChemExpress.
Zhao Z., Shang X., Chen Y., Zheng Y., Huang W., Jiang H., Lv Q., Kong D., Jiang Y, & Liu P. (2020). Bacteria elevate extracellular adenosine to exploit host signaling for blood-brain barrier disruption. Virulence, 11(1), 980-994.
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6

Characterization of Signaling Pathways

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Isoproterenol, formoterol, GO6983, GO6976, RO32–0432, GF109203X, H89, PSB603, ESI-09, TPPB, NECA (adenosine-5′-N-ethyluronamide), MRS5698 and MRS2365 ([[(1R,2R,3S,4R,5S)-4-[6-amino-2-(methylthio)-9H-purin-9-yl]-2,3-dihydroxybicyclo[3.1.0]hex-1-yl]methyl] diphosphoric acid monoester trisodium salt) were purchased from Tocris (Ellisville, MO). CGS21680">CGS21680, carbachol, cholera toxin (CTX) and PTX were from Sigma (St. Louis, MO). YM254890, enzastaurin, LY333531, VTX-27 and AB928 were purchased from MedChemExpress (Monmouth Junction, NJ, USA). BAY60-6583 (LUF6210, termed hereafter ‘BAY’) was synthesized at Leiden/Amsterdam Center for Drug Research and was provided by Ad IJzerman (Leiden, The Netherlands). All compounds were dissolved in DMSO except that CTX and PTX were in water, and proper controls were included in all experiments. AlphaScreen cAMP kit, SureFire p-ERK1/2 (Thr202/Tyr204) Assay Kit and AlphaScreen SureFire p-Akt 1/2/3 (p-Ser473) Assay Kit were purchased from PerkinElmer (Waltham, MA). Gs-null and Gq/11-null HEK293 cells were generated at Tohoku University, Sendai, Japan. HEK293 human embryonic kidney, PC-3 human prostate cancer, NIH-3T3 mouse fibroblast, and H9C2 rat cardiomyoblast cells were from ATCC (Manassas, VA); all other reagents were from standard commercial sources and of analytical grade.
Gao Z.G., Levitan I.M., Inoue A., Wei Q, & Jacobson K.A. (2023). A2B adenosine receptor activation and modulation by protein kinase C. iScience, 26(7), 107178.
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7

Adipogenic Differentiation of hMSCs

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The hMSCs (Institute for Regenerative Medicine, Texas A&M University) were maintained in growth medium containing high-glucose DMEM, 10% (v/v) fetal bovine serum (FBS; HyClone), 4 mM L-glutamine, and 50 U/mL penicillin/ streptomycin. The cells were passaged at approximately 70% confluence.
Prior to seeding hMSCs, sterile nonmineralized and mineralized hydrogels as well as cell culture-grade coverslips (CS; diameter = 15 mm, Fisherbrand, catalog no. 1254582) were incubated in growth medium at 37 °C for 24 h to promote cell adhesion. Passage 5 (P5) hMSCs were plated at a density of 12 000 cells/cm2 and cultured for 14 days using either growth medium or adipogenic medium at 37 °C and 5% CO2, with medium change every 2 days. Adipogenic medium was prepared by supplementing growth medium with 1 μM dexamethasone (Sigma-Aldrich, catalog no. D2915), 200 μM indomethacin (Sigma-Aldrich, catalog no. I7378), 0.5 μM 3-isobutyl-1-methylxanthine (IBMX; Sigma-Aldrich, catalog no. I5879), and 10 μg/mL of insulin (Sigma-Aldrich, insulin from bovine pancreas, catalog no. I6634). For pharmacological inhibition of A2bR, PSB 603 (Tocris Bioscience, catalog no. 3198) was added into growth medium or adipogenic medium at varying concentrations of 0, 0.5, 10, and 100 nM.
Kang H., Shih Y.R, & Varghese S. (2015). Biomineralized Matrices Dominate Soluble Cues To Direct Osteogenic Differentiation of Human Mesenchymal Stem Cells through Adenosine Signaling. Biomacromolecules, 16(3), 1050-1061.
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8

Adenosine Receptor Modulation Experiments

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The following ARs agonists and antagonists were purchased from Tocris (Bristol, UK): NECA (5′-(N-Ethyl-carboxamido)adenosine); CGS15943 (9-Chloro-2-(2-furanyl)-[1,2,4]triazolo[1,5-c]quinazolin-5-amine); DPCPX (1,3-Dipropyl-8-cyclopentylxanthine); SCH442416 (2-(2-Furanyl)-7-[3-(4-methoxy-phenyl)propyl]-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine); PSB603 (8-(4-(4-(4-Chlorophenyl)piperazide-1-sulfonyl) phenyl)-1-propylxanthine). CCPA (2-Chloro-N6-cyclopentyladenosine) and MRS1523 (3-Propyl-6-ethyl-5-[(ethylthio)carbonyl]-2 phenyl-4-propyl-3-pyridine carboxylate), adenosine deaminase (ADA), l-glutamic acid monosodium salt monohydrate, forskolin, KT5720, tert-Butyl hydroperoxide (TBHP), N-acetylcysteine (NAC) were obtained from Sigma-Aldrich (St. Luis, MO, USA). Annexin V AlexaFluorTM 488 Ready Flow Conjugate, SYTOX™ AADvanced™ Dead Cell Stain, CellROX™ Green Reagent, MitoTracker™ Red CMXRos were purchased from Life Technologies (Monza, Italy). The A1AR positive allosteric modulator TRR469 was previously synthesized and characterized (compound 4ad in [23 (link)]).
Vincenzi F., Pasquini S., Gessi S., Merighi S., Romagnoli R., Borea P.A, & Varani K. (2020). The Detrimental Action of Adenosine on Glutamate-Induced Cytotoxicity in PC12 Cells Can Be Shifted towards a Neuroprotective Role through A1AR Positive Allosteric Modulation. Cells, 9(5), 1242.
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9

Modulation of Schizophrenia-like Behaviors

Cited 1 time
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A total of 40 mice were randomly divided into four groups (10 mice per group) as follows: Control group (Control), MK-801 group (MK-801), MK-801 plus A2BAR agonist group (MK-801 + BAY) and MK-801 plus A2B AR antagonist group (MK-801 + PSB). The SCZ model was established according to a previously described method (26 (link)). Briefly, mice were injected intraperitoneally with MK-801 (0.6 mg/kg/day; cat. no. M107; MilliporeSigma) once a day for 14 consecutive days, and the intervention was performed between 3:00 and 4:00 p.m. every day. The control group was treated with the same volume of normal saline. The optimal dose of A2BAR selective agonist BAY 60-6583 (21 (link),27 (link),28 (link)) and A2B AR selective antagonist PSB 603 (21 (link),29 (link)) was determined according to previous studies. Intraperitoneal injection of BAY 60-6583 (80 µg/kg/day; cat. no. 910487-58-0; Tocris Bioscience) and PSB 603 (25 µg/kg/day; cat. no. 1092351-10-40; Tocris Bioscience) was administered to the MK-801 + BAY and MK-801+ PSB groups, respectively, every 2 days between day 8 and day 14 of the MK-801 treatment period. The mice in the control and MK-801 groups were intraperitoneally injected with an equal volume of normal saline.
Ma Q., Wang D., Li Y., Yang H., Li Y., Wang J., Li J., Sun J, & Liu J. (2022). Activation of A2B adenosine receptor protects against demyelination in a mouse model of schizophrenia. Experimental and Therapeutic Medicine, 23(6), 396.
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10

Adenosine Receptor Inhibition Assay

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A2AR-HEK cells were grown for 24 hrs in 96 well plates. The medium was then removed, and the cells washed twice with PBS. Cells were then treated with assay buffer containing the A2BR antagonist PSB603 (1 µM, Tocris), rolipram (30 µM, Sigma) and adenosine deaminase (3 U/ml, Worthington) for 30 min followed by addition of agonists or vehicle control and incubated for 20 min. The reaction was terminated by removal of the mixture of assay buffer and compounds, and addition of 50 µl 0.3% Tween-20. cAMP levels were measured with an Amplified Luminescent Proximity Homogeneous Assay (ALPHA) Screen cAMP assay kit as instructed by the manufacturer (PerkinElmer, Waltham, MA).
Schwarz A.M., Keresztes A., Bui T., Hecksel R.J., Peña A., Lent B., Gao Z.G., Gamez-Rivera M., Seekins C.A., Chou K., Appel T.L., Jacobson K.A., Al-Obeidi F.A, & Streicher J.M. (2023). Terpenes from Cannabis sativa Induce Antinociception in Mouse Chronic Neuropathic Pain via Activation of Spinal Cord Adenosine A2A Receptors. bioRxiv.
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