The chemical compound library was from Open Innovation Center for Drug Discovery, University of Tokyo (Tokyo, Japan) (23 ). The library includes 174,131 compounds supplied as 10 or 2 mM solutions in DMSO. After the selection of N-phenylmaleimide derivatives, each compound was obtained in powdered form from commercial vendors (see supplementary Methods). Methylcarbamyl-PAF (mcPAF, nonhydrolyzed analog of PAF), 16:0 PAF, 16:0 lyso-PAF, deuterium-labeled (d4)-16:0 PAF, d4-16:0 lyso-PAF, and d31-16:0 lyso-PC were from Cayman Chemical Company (Ann Arbor, MI). DPPC standards were purchased from NOF Corporation (Tokyo, Japan). Palmitoyl-CoA and arachidonoyl-CoA were from Avanti Polar Lipids (Alabaster, AL). Acetyl-CoA, DMSO, chloroform, and LC-MS grade solvents (methanol and acetonitrile) were from Wako (Osaka, Japan). Lipopolysaccharide (LPS) from Salmonella minnesota was purchased from Sigma-Aldrich (St. Louis, MO), and A23187 (calcium ionophore) was from Biomol (Plymouth Meeting, PA). Two types of protease inhibitor cocktails (complete and EDTA-free complete) were purchased from Roche Applied Science (Mannheim, Germany).
Arachidonoyl coa
Manufactured by Avanti Polar Lipids
Arachidonoyl-CoA is a coenzyme A derivative of the polyunsaturated fatty acid arachidonic acid. It is an important intermediate in the biosynthesis of eicosanoids, a class of signaling molecules involved in various physiological and pathological processes.
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Lab products found in correlation
2 protocols using arachidonoyl coa
1
Characterization of Lipid Compounds
2
Measuring Lpcat3 Activity in Adipocytes
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After 6 days of differentiation and 2 days of adenovirus infection, the adipocytes were collected and resuspended in enzyme reaction buffer (75 mM Tris, 1 mg/mL BSA, pH 6.0). The cell membrane was disrupted by ultrasound and then centrifuged at 10,000
g. The supernatant was collected to determine the protein concentration. Lpcat3 activity assays were performed as previously described
[29] (link). Briefly, the reaction mixture in a total volume of 100 μL containing 20 μg protein, 11 μM NBD-Lyso-PC (Avanti® Polar Lipids), and 11 μM arachidonoyl-CoA (Avanti® Polar Lipids) was incubated for 10 min at 30°C. The reaction was stopped by the addition of 300 μL of chloroform/methanol (1:1; v/v) and vigorous mixing. Then, the lower organic phase was collected after centrifugation (6000
g) and dried under nitrogen gas. Lipids were redissolved and then separated with a TLC plate. The development solvent for TLC was chloroform/methanol/water (65:25:4; v/v). Bands were detected under UV, and the product content was determined using ImageJ software.
g. The supernatant was collected to determine the protein concentration. Lpcat3 activity assays were performed as previously described
[29] (link). Briefly, the reaction mixture in a total volume of 100 μL containing 20 μg protein, 11 μM NBD-Lyso-PC (Avanti® Polar Lipids), and 11 μM arachidonoyl-CoA (Avanti® Polar Lipids) was incubated for 10 min at 30°C. The reaction was stopped by the addition of 300 μL of chloroform/methanol (1:1; v/v) and vigorous mixing. Then, the lower organic phase was collected after centrifugation (6000
g) and dried under nitrogen gas. Lipids were redissolved and then separated with a TLC plate. The development solvent for TLC was chloroform/methanol/water (65:25:4; v/v). Bands were detected under UV, and the product content was determined using ImageJ software.
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