Rb1 (>98%, ab142646) was purchased from Abcam (Cambridge, UK). 3-Isobutyl-1-methylxanthine (IBMX), dexamethasone (Dex), insulin, and Oil Red O powder were purchased from Sigma (St. Louis, MO, United States). L748337 was from Tocris Bioscience (Bristol, UK). Dulbecco’s Modified Eagle’s Medium (DMEM) and fetal bovine serum (FBS) were purchased from Corning (NY, United States). Antibodies for liver kinase B1 (LKB1) (3047S), pLKB1 (Ser428) (3482S), AMP-activated protein kinase alpha (AMPKα) (2532S), pAMPKα (Thr172) (2535S), acetyl-CoA carboxylase (ACC) (3676S), pACC (Ser79) (3661S), silent information regulator T1 (SIRT1) (8469S), SIRT3 (5490S), phospho-hormone sensitive lipase (pHSL) (Ser563) (4139S), phospho-PKA substrate (9624S), UCP1 (14670S), and β-actin (3700S) were purchased from Cell Signaling Technology (Beverly, MA, United States); the antibody for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (sc-32233) antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, United States); antibodies for PGC1α (ab54481), comparative gene identification 58 (CGI58) (ab59488), adipose triglyceride lipase (ATGL) (ab207799), HSL (ab45422), and β3AR (ab94506) were purchased from Abcam (Cambridge, UK); the antibody for PPARα (PA1-822A) was purchased from Thermo Fisher Scientific (Waltham, MA, USA).
L748337
Manufactured by Bio-Techne
Sourced in United Kingdom, United States, Germany
L748337 is a laboratory instrument designed for use in biological research and analysis. It is a compact, self-contained unit capable of performing a variety of tasks related to sample preparation, processing, and analysis. The core function of this product is to provide researchers with a versatile and reliable tool for their laboratory workflows.
Automatically generated - may contain errors
Lab products found in correlation
Isoproterenol, by Merck Group (2 mentions)
Nadolol, by Merck Group (2 mentions)
Doxazosin mesylate, by Bio-Techne (2 mentions)
Ab207799, by Abcam (1 mentions)
Ab45422, by Abcam (1 mentions)
Ab94506, by Abcam (1 mentions)
Ab54481, by Abcam (1 mentions)
β actin 3700, by Cell Signaling Technology (1 mentions)
Oil red o powder, by Merck Group (1 mentions)
Dulbecco s modified eagle s medium dmem, by Corning (1 mentions)
Fetal bovine serum (fbs), by Corning (1 mentions)
Bkm120, by Selleck Chemicals (1 mentions)
L name, by Merck Group (1 mentions)
Lapatinib, by LC Laboratories (1 mentions)
Herceptin, by Genentech (1 mentions)
Dorsomorphin, by Abcam (1 mentions)
Brl 37344, by Bio-Techne (1 mentions)
Trans cinnamic acid, by Merck Group (1 mentions)
Prism 6, by GraphPad (1 mentions)
Nebivolol, by Bio-Techne (1 mentions)
8 protocols using l748337
1
Molecular Mechanisms of Adipocyte Metabolism
2
High-throughput Drug Screening for Cancer Therapy
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Trastuzumab (Herceptin®, manufactured by Genentech, San Francisco, CA, United States) was purchased from McKesson and was dissolved in sterile, distilled water provided with the drug. Lapatinib was obtained from LC Laboratories (MA, United States) and was dissolved in sterile dimethyl sulfoxide (DMSO). For the screening, a library of 284 drugs/compounds was purchased from Selleck laboratories. Additional 38 FDA-approved drugs were purchased from Tocris Bioscience, 20 were purchased from MedChemExpress, and 8 were from Sigma (Supplementary File S1 ). Drug dilutions were made in appropriate media such that the final DMSO concentration was less than 0.1%. Anisomysin and BKM-120 were purchased from Selleck and were used as positive controls. For validation studies, nebivolol, carvedilol and metoprolol were purchased from Selleck laboratories, L-748337 was purchased from Tocris Bioscience, and L-NAME was purchased from MilliporeSigma.
3
Characterization of cinnamic acid
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Trans-cinnamic acid (99% purity, Figure 1 A) was purchased from Sigma Chemical Co. (St. Louis, MO, USA). BRL 37344 and L-748.337 were purchased from Tocris Bioscience (Bristol, UK). AICAR was purchased from TCI (Chuo-ku, Tokyo, Japan). Dorsomorphin was purchased from Abcam (Cambridge, UK). All other chemicals used in this study were of analytical grade.
4
Radioligand Binding Assay for β3 Receptor
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Membrane preparations ranged between 0.25–2 µg of protein/well. The radioligand binding experiments were done in a volume of 200 µl (50 µl Hanks’ Balanced Salt Solution (HBSS) assay buffer, 20 mM HEPES, 0.1% BSA, pH 7.4; 25 µl antagonist or assay buffer (depending on assay type), 50 µl membrane solution (final protein concentration 5 µg), 50 µl scintillation proximity assay (SPA) solution (Perkin Elmer), and 25 µl of dihydroalprenolol hydrochloride, levo-[ring, propyl-3H(N)] (PerkinElmer). For saturation binding experiments, we used up to 300 nM of [3H]-dihydroalprenolol. Different dilutions of the radioligand were prepared in assay buffer corresponding to a concentration range of approximately 0.02–300 nM. Non-specific binding was determined in the presence of 10 µM of the selective β3 antagonist L748337 (TOCRIS). Samples were incubated in 96-well plate sealed with transparent Topseal for 2 h at 25 °C with gentle agitation. Samples were centrifuged for 10 min at 2500g before being analyzed in a β-counter. Data were fitted using a non-linear regression, one site binding model with GraphPad Prism 6.0 (GraphPad Software, Inc.).
5
Receptor Binding Assay Protocol
6
Vascular Contractility Assays with Pharmacological Agents
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U46619 (Tocris Bioscience, Bristol, United Kingdom), mirabegron (Tocris Bioscience), doxazosin mesylate (Tocris Bioscience), nadolol (Sigma-Aldrich, St. Louis, MO, USA), L-748,337 (Tocris Bioscience) and bupranolol (Toronto Research Chemicals, Toronto, Canada) were dissolved in dimethyl sulfoxide. Isoproterenol hydrochloride (Sigma-Aldrich), BK (Sigma-Aldrich), clonidine (Sigma-Aldrich), yohimbine (Sigma-Aldrich), L-NMMA (Sigma-Aldrich), ET-1 (Calbiochem, St. Louis, MO, USA) and PE (Sigma-Aldrich) were dissolved in distilled H 2 O. Physiological salt solution (PSS) contained (mmol L -1 ): NaCl 115; NaHCO 3 25; K 2 HPO 4 2.5; MgSO 4 1.2; glucose 5.5; HEPES 10; and CaCl 2 1.3 (pH 7.4). High-K + solution was obtained as a mix of PSS and a solution containing NaCl 20 mmol L -1 and KCl 95 mmol L -1 , to obtain a K + concentration of 32 mmol L -1 in the organ chamber. Buffers were continuously aerated with 5% CO 2 in air at 37 • C. All the indicated concentrations of the pharmacological agents added to the bathing solution represent their final concentration in the organ chamber.
7
Adrenoceptor Pharmacology Toolbox
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Phenylephrine hydrochloride (selective α 1 AR agonist), dexmedetomidine hydrochloride (selective α 2 AR agonist), L-748, 337 (β 3 AR antagonist), doxazosin mesylate (α 1 /α 2 AR antagonist) and RS79948 hydrochloride (selective α 2 AR antagonist) were obtained from Tocris/Bio-Techne (Wiesbaden, Germany).
Isoproterenol (unselective β-AR agonist), norepinephrine (unselective AR agonist), BRL 37344 (selective β 3 -AR agonist) and nadolol (non-selective β AR agonist) were obtained from Sigma Aldrich (St. Louis, USA).
Isoproterenol (unselective β-AR agonist), norepinephrine (unselective AR agonist), BRL 37344 (selective β 3 -AR agonist) and nadolol (non-selective β AR agonist) were obtained from Sigma Aldrich (St. Louis, USA).
8
Membrane Protein Extraction and Binding
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For membrane preparation, cells were thawed on ice for 30min. The cells were lysed in 20mM Tris, pH 8, 500mM NaCl, 15% glycerol, 50mg/L DNAse I, 1 tablet/5L cell suspension of the EDTA-Free cOmplete Protease Inhibitor Cocktail (Roche) and 20M carvedilol using a continuous flow EmulsiFlex-C3 cell disruptor (Avestin). The cell lysate was centrifuged at 10'000g for 30min at 4C, followed by centrifugation of the resulting supernatant at 100'000g for 1h at 4C. The membranes were flash-frozen in liquid nitrogen and stored at -80C for further use. For saturation binding experiments, we used up to 300nM of [3H]-dihydroalprenolol.
Different dilutions of the radioligand were prepared in assay buffer corresponding to a concentration range of approximately 0.02-300nM. Non-specific binding was determined in the presence of 10µM of the selective β3 antagonist L748337 (TOCRIS). Samples were incubated in 96-well plate sealed with transparent Topseal for 2h at 25°C with gentle agitation. Samples were centrifuged for 10min at 2'500g before being analyzed in a -counter. Data were fitted as one-site binding using Prism.
Statistical analyses were performed with Prism (GraphPad) using the unpaired t test.
Different dilutions of the radioligand were prepared in assay buffer corresponding to a concentration range of approximately 0.02-300nM. Non-specific binding was determined in the presence of 10µM of the selective β3 antagonist L748337 (TOCRIS). Samples were incubated in 96-well plate sealed with transparent Topseal for 2h at 25°C with gentle agitation. Samples were centrifuged for 10min at 2'500g before being analyzed in a -counter. Data were fitted as one-site binding using Prism.
Statistical analyses were performed with Prism (GraphPad) using the unpaired t test.
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