ADP-Glo kinase assay

The ADP-Glo kinase assay (Promega, Madison, WI) protocol was adapted from previous protocols (Baskaran et al., 2014 ) and performed in 96-well PS F-Bottom white plates (Greiner Bio-One, Frickenhausen, Germany). The 25-μl reaction consisted of 20 μl of 125 μM PI: phosphatidylserine (PS) (Avanti Polar Lipids, Alabaster, AL) SUVs in PI/PS buffer (50 mM HEPES, pH 7.0, 50 mM MnCl₂, 0.5 mM TCEP), and 2.5 μl of 100 nM recombinant PI3KC3 in PI3K freezing buffer and was initiated by adding 2.5 μl of 500 mM ATP. After incubation for 1 h at 37°C, the reaction was stopped by the addition of 25 μl ADP-Glo Reagent supplemented with 10 mM MgCl₂ for 40 min at 25°C. Then, 50 μl of Kinase Detection Reagent was added to wells and incubated for 30 min at 25°C. Luminescence was measured using an Infinite M1000 system (Tecan, Research Triangle Park, NC) with a 1000-ms integration time.