Assay buffer 1

For phosphorylation reactions, predetermined concentrations of protein kinase and PI3K complexes were added to master mix containing 50 mM of ATP, 0.1 mg/mL of BSA and 1x Assay Buffer I (SignalChem) in 40 mL total volumes. Reactions were carried out at 30°C for 30 min. Next, 0.01 mCi/mL ³²P-labeled ATP (PerkinElmer) and 25 μM PIP₂ (Thermo Scientific) were included, bringing total volume to 100 mL PIP₂ phosphorylation reactions were carried out at 30°C for 20 min 50 μl of 4N HCl was added to quench the reaction and the lipid was extracted with 100 mL of CHCl3: MeOH (1:1) followed by vortexing for 1 min and centrifugation at maximum speed for 2 min. 10 mL of the lower hydrophobic phase was extracted with gel loading pipet tips and spotted onto a silica plate (EMD Millipore #M116487001) for thin-layer chromatography. Plates were placed in a sealed chamber with 1-propanol: 2M acetic acid (65:35). Radioactivity was imaged with a Typhoon FLA 7000 phosphorimager (GE) and quantified by ImageQuant (GE). For cationic liposomes, EPC (#890704C) and PI (840042C) were purchased from Avanti. Liposomes containing 5% PI, 60% EPC, and 35% cholesterol were prepared by extrusion via 20 passes through a 0.1 mm membrane using a Mini-Extruder kit (Avanti). The anionic liposomes in these experiments replaced EPC with PS.

Cell were lysed using the PI3K buffer [25 mM Tris, pH 7.5, 10 mM EDTA, 10 mM EGTA, 1% Nonidet P-40] with one tablet (per 10 mL) of protease and phosphatase inhibitor (Life Technologies). After lysis on ice for 30 min, cell lysates were centrifuged for 10 min, 4°C and supernatant were used for immunoprecipitation. Lysates were incubated with anti-phospho-Tyr-4G10 (EMD Millipore 05–321), anti-p110a (CST #4249) or anti-p85a antibody (EMD Millipore #ABS233) for 1 h, followed by protein A/G beads (Santa Cruz) for additional 1hr, and then washed twice with PI3K buffer, and three times with TNE buffer [10 mM Tris (pH 7.5), 100 mM NaCl, 1 mM EDTA]. For PI3K activity assays, the beads (50 μL) were incubated in 1′ Assay Buffer I (SignalChem), 0.5 μL ATP (10 mM), 1 μL ³²P-labeled ATP (0.01 mCi/mL) and 10 μL of PIP₂ (250 μM) in 100 μL total volumes. Reactions were carried out at 30°C for 30 min 50 μl of 4N HCl was added to quench the reaction and the lipid was extracted with 100 mL of CHCl3: MeOH (1:1) followed by vortexing and centrifugation. 20 mL of the lower hydrophobic phase was extracted with gel loading pipet tips and spotted onto a silica plate (EMD Millipore, M116487001) for thin-layer chromatography. Plates were placed in a sealed chamber with 1-propanol: 2M acetic acid (65:35). Radioactivity was visualized and quantified using a Typhoon FLA 7000 phosphorimager.