BI01383298, 1-(3,5-Dichlorophenylsulfonyl)-N-(4-fluorobenzyl)piperidine-4-carboxamide, was purchased from Medkoo Biosciences (Chapel Hill, NC, U.S.A.). BI01372674, chemically analogous to BI01383298, was provided by Boehringer Ingelheim Pharma GmbH (Ingelheim, Germany). PF06761281, (2R)-2-Hydroxy-2-[2-(2-methoxy-5-methyl-3-pyridinyl)ethyl]succinic acid, was kindly supplied by Pfizer Inc. (New York, NY, U.S.A.). [14C]-Citrate (specific radioactivity, 113 mCi/mmol) and [14C]-nicotinate (specific radioactivity, 50 mCi/mmol) were purchased from Moravek Biochemicals (Brea, CA, U.S.A.). [3H]-Arginine monohydrochloride (specific radioactivity, 54.5 mCi/mmol), [14C]-lactate (specific radioactivity, 154.8 mCi/mmol), [3H]-glutamine (specific radioactivity, 50.5 mCi/mmol), and [3H]-tryptophan (specific radioactivity, 20.1 mCi/mmol) were purchased from PerkinElmer (Waltham, MA, U.S.A.). All other reagents were from Millipore-Sigma (St. Louis, MO, U.S.A.) or Thermo Fisher Scientific (Waltham, MA, U.S.A.).
14c lactate
Manufactured by PerkinElmer
Sourced in United States
14C-lactate is a radiolabeled compound that contains the radioactive carbon isotope 14C. It is a commonly used tracer molecule in scientific research and clinical applications. The primary function of 14C-lactate is to facilitate the study of metabolic processes and pathways involving lactate, a key intermediate in cellular energy production.
Automatically generated - may contain errors
4 protocols using 14c lactate
1
Synthesis of Novel Sulfonylpiperidine Compounds
2
Measuring PHD2 Enzyme Kinetics
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14C-lactate (5μCi/ml) (PerkinElmer) with or without unlabelled lactate was incubated with 25 μg/ml PHD2181-426 protein in the reaction buffer (20 mM Tris pH = 7.5, 5 mM KCl, 1.5 mM MgCl2, 1 mM DTT, 2 mM ascorbate, 100 μM FeSO4) at 37 °C for 1 hr. His-tagged PHD2181-426 was pulled down by Ni-NTA beads and the radioactivity of the beads was measured with liquid scintillation counter.
3
Comprehensive Cellular Metabolite Analysis
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AR-C 155858 (AR-C) was from AdooQ Bioscience, α-Cyano-4-hydroxycinnamic acid (4-CIN) and isoproterenol from Sigma-Aldrich, and14C-lactate from PerkinElmer. Antibodies against MCT1 and MCT4 were from Millipore, and antibodies against carbonic anhydrase II (CAII) and NBCe1 from Abcam. Antibodies against p150Glued and transcription factor IIB (TFIIB) were from BD Biosciences and Santa Cruz Biotechnology, respectively. Unless otherwise mentioned, other reagents were purchased from Sigma-Aldrich. Antibodies against hormone-sensitive lipase (HSL), phospho-Ser660-HSL, phospho-Ser133 cAMP-responsive element binding protein (CREB), poly-ADP-ribose (PARP), and phospho-Thr180/Tyr182-p38 MAPK were from Cell Signaling Technology, Leiden, The Netherlands.
4
Quantification of Cellular Lactate Uptake
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On day 2 after siRNA transfection, KMM-1 cells were incubated in uptake buffer (10 mM HEPES, 5 mM KCl, 150 mM NaCl, 1 mM MgCl2, pH 7.5) containing 14C-lactate (PerkinElmer, Waltham, MA, USA) for 1 h at 37°C. Uptake was stopped by incubating the cells at 4°C, and the cells were washed twice using 200 μL of uptake buffer. The cells were then lysed in 200 μL of uptake buffer containing 1% SDS and the cell lysates were collected into scintillation vials for quantification of 14C-lactate uptake. To ensure selective incorporation of lactate, some cells were simultaneously treated with 14C-lactate and unlabeled lactate (10 mM).
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