Differential sensitivity of phosphatidylinositol 3-kinase p110gamma to isoforms of G protein betagamma dimers. The overall rank order of inhibitors was the same using the C8 and C16 substrates, except for minor deviations. ATP hydrolysis in the absence of substrate was detected with the PI3K isoform, and inhibitors affected PI3K intrinsic ATP hydrolysis activity similarly to lipid Pluripotin (SC-1) phosphorylation. concentrations of: 50 mM HEPES (pH 7.5), 200 mM NaCl, 10 mM EDTA, 0.01% Brij-35, 2 nM ADP AlexaFluor? 633 tracer, and 15.5 g/ml ADP antibody. The concentration of ADP antibody used was equal to the EC85 concentration in the presence of 30 M ATP, the concentration of ATP used in all kinase reactions. Fluorescence polarization measurements were performed on a Tecan Ultra plate reader using the following filters and settings: 612 nm excitation filter (10 nm bandwidth), 670 nm emission filter (25 nm bandwidth), 10 flashes per well, 30C, or around the Tecan Safire2? plate reader using the following filters and settings: 635 nm excitation (LED), 670 nm emission (10 nm bandwidth), 10 flashes per well, 30C. A free tracer reference was set to 20 mP, and the buffer (made up of ADP antibody) was used as the buffer blank for both the sample and free tracer reference wells. TR-FRET Detection For TR-FRET detection, PI3K reactions were stopped by the addition of an equal volume (10 L) of detection mix to Pluripotin (SC-1) yield concentrations of: 50 mM HEPES (pH 7.5), 100 mM Pluripotin (SC-1) NaCl, 5 mM EDTA, 0.01% Brij-35, 2 nM ADP antibody-Tb, and 14 nM ADP FAM tracer. The concentration of ADP FAM tracer used was equal to the EC50 concentration in the presence KLF4 of 30 M ATP in the kinase enzyme reaction. TR-FRET measurements were performed around the Tecan Ultra plate reader (Durham, NC) using the following filters and settings: 340 nm excitation filter (35 nm bandwidth), 495 nm (10 nm bandwidth) and 520 nm (25 nm bandwidth) emission filters, 100 sec delay, 100 sec integration time, 10 flashes at 30C. Lipid Substrate Vesicle Preparation Lipid vesicles were prepared by sonication, freeze/thaw, or a combination of the two methods. The phosphatidylinositol 4,5 bisphosphate (PI(4,5)P2) substrate with fatty acid side-chains of eight (C8) or sixteen (C16) carbons were suspended in water to a concentration of 1310 M and 910 M, respectively. In addition, an aliquot of the PI(4,5)P2 C16 sample was removed Pluripotin (SC-1) and an equimolar concentration of phosphatidylserine (PS) was added prior to sonication. Bath sonication was performed at 50/60 Hz/80 watts/117 volts for 1 hour at 27C33C. In addition, aliquots from the sonicated PI(4,5)P2 C16 lipid substrate preparation were removed and frozen and thawed 5 occasions. The samples were frozen in an isopropanol/dry ice bath, with thawing in a water bath at 40C and vigorous vortexing. Long chain fatty acids stick to plastic. Therefore, all manipulations of the PI(4,5)P2 C16 lipid substrate were performed in glass vials. Long term storage for lipid substrates was at ?80C. ADP/ATP Standard Curve 12-point ADP/ATP standard curves designed to mimic an enzyme reaction were used to quantify ADP production in the PI3K enzyme reactions. Starting at 30 M ATP – the concentration used in PI3K reactions – ATP was decreased and ADP increased proportionately, keeping the total adenosine concentration constant. The standard curves (n = 4) contained all of the components used in the authentic enzyme assays except enzyme, and were included on the same plates as the experimental reactions. Based on the standard curves for both TR-FRET and FP readouts, the concentration of ADP produced in the enzyme reactions was calculated using the Graphpad PRISM software using the four-parameter logistic regression curve fit. Because there are alternate ways to fit data to a non-linear standard curve, we validated the goodness of fit using the backcalculation method  and individual data points within an ADP/ATP standard curve. To minimize error propagation from the highest and lowest regions of the standard curves, enzyme reactions were designed so that the amount of ADP produced (in the absence of inhibitor) fell mostly within the middle region of the curves. Inhibitor titrations Dose dependency is shown for each inhibitor from a 20-point two-fold dilution.