PI3K kinase activities

A single-molecule kinase assay was employed to quantify PI3Kα lipid kinase activity and PIP₃ production, as previously described (19). The method counts all single molecules of product PIP₃ generated by the PI3K lipid kinase reaction, using a saturating concentration of fluor-tagged, GRP-PH domain (800 pM) to bind and detect each PIP₃ molecule generated on the membrane surface. After PI3K was added to the chamber (see Single-Molecule TIRFM Measurements above), ATP (1 mM) was added from a buffered stock (assay buffer containing 100 mM ATP and 82.5 mM Mg²⁺) to start the kinase reaction. Subsequently, fluor-tagged PH domain tracks were quantified as previously described at five time points (18, 19). To determine the PI3Kα specific activity, the resulting net rate of PIP₃ production is divided by the average density of PI3Kα determined by the above binding assay (with appropriate correction for the PI3K fluorescence labeling efficiency).