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DAG measurement at the plasma membrane and in the cytosol

JB Julien Bricambert
MA Marie-Clotilde Alves-Guerra
PE Pauline Esteves
CP Carina Prip-Buus
JB Justine Bertrand-Michel
HG Hervé Guillou
CC Christopher J. Chang
MW Mark N. Vander Wal
FC François Canonne-Hergaux
PM Philippe Mathurin
VR Violeta Raverdy
FP François Pattou
JG Jean Girard
CP Catherine Postic
RD Renaud Dentin
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Hepatic DAG content was separated into membrane and cytoplasmic fractions as previously described60. Briefly, liver samples (250 mg) were homogenized on ice using a dounce homogenizer in lysis buffer containing 10 mM Tris base, 0.5 mM EDTA, 250 mM sucrose, and protease inhibitors. Then 3% sucrose was layered on top of the homogenate, and samples were centrifuged at 100,000g for 1 h at 4 °C. The supernatant and lipid layers were removed and designated as the cytoplasmic fraction. The pellet, designated as the membrane fraction, was resuspended in homogenization buffer for DAG analysis. Concentrations of DAG in each fraction were determined by using mass spectrometry analysis as previously described.

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