In vitro kinase assay

For phosphorylation reactions, predetermined concentrations of protein kinase and lipid kinases were added to master mix containing 50 μM of ATP (Sigma), 0.1 mg/mL of BSA and 1x Assay Buffer I (SignalChem) in 40 μl total volumes. Reactions were carried out at 30°C for 30 minutes. Next, 0.01 mCi/mL ³²P-labeled ATP (Perkin Elmer) and 25 μM PI5P:PS or PI4P:PS liposomes (Avanti) were included in the kinase reaction, bringing total volume to 100 μL. Additional cold ATP was included to maintain its concentration at 50 μM. Lipid phosphorylation reactions were carried out at room temperature for 20 min. 50 μl of 4N HCl was added to quench the reaction and the lipid was extracted with 100 μl of CHCl3: MeOH (1:1) followed by vortexing for 1 minute and centrifugation at 14,000XG for 2 minutes. 10 μL 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) mobile phase. After completion, the plates were removed, briefly dried, exposed to a storage phosphor screen BAS-IP (GE Life Sciences) and imaged with a Typhoon FLA 7000 phosphorimager (GE) and quantified by ImageQuant (GE). PI5P:PS or PI4P:PS liposomes were prepared by mixing lipid substrate with 2-fold mass excess PS in chloroform. The chloroform was evaporating with N₂ gas, and the lipid mixture was hydrated in 30 mM HEPES pH 7.4, 1 mM EGTA and dispersed by bath sonication.