DAG Kinase Assay

The DAG kinase assay protocol was adapted from procedures described previously⁴². Cells growing in 100mm cell culture dishes at 80% confluency were lysed on ice using the following lysis buffer: 50 mM HEPES, pH 7.2, 150 mM NaCl, 5 mM MgCl2, 1 mM dithiothreitol, 1 mM phosphatase inhibitor cocktail and 1mM protease inhibitor cocktail. After centrifugation at 400 g for 5 min, the resultant supernatant was used for the DAG Kinase activity assay. The enzymatic reactions were carried out in triplicate in 384-well white plates, in the final volume of 10uL, in the solution of the following final composition: 50 mM MOPS, pH 7.4, 50 mM n-octyl b-D-glucopyranose (Sigma-Aldrich), 1 mM dithiothreitol, 100 mM NaCl, 20 mM NaF, 10 mM MgCl₂, 1 mM CaCl₂, 10 mM phosphatidylserine (Sigma-Aldrich), 2 mM 1,2-dioleoyl-sn-glycerol (Sigma-Aldrich), 0.2 mM ATP. The enzymatic reactions were incubated at 37°C for 90 minutes. 10uL of ADP-Glo reagent (Promega) was added at 25°C. Following 40 minutes incubation, 20uL of Kinase Detection Reagent (Promega) was added. After additional 40 minutes of incubation at 25 °C, luminescence was detected using the BioTek plate reader (BioTek, Winooski, VT, USA) with sensitivity set to 100. ATP was used as a positive control and lysates heated at 70°C for 15 minutes (protein denaturing conditions) were used as a negative control.