In vitro Vps34 Lipid Kinase Assay

In vitro Vps34 lipid kinase assay was performed as previously described with minor modification^{18 (link)}. Briefly, Vps34 immune-complex beads were washed once with kinase assay (KA) buffer (20 mM HEPES, pH7.4, 1 mM EGTA, 0.4 mM EDTA and 5 mM MgCl₂), then suspended in 50 μl KA buffer containing 50 μM cold ATP, 5 mM MnCl₂, 50 μM DTT, 0.1 mg/ml Sonicated phosphatidylinositol and 5 μCi ³²P-ATP. The mixtures were incubated at 37°C for 30 minutes with vigorous shaking. The reactions were stopped by adding 120 μl of CHCl3/CH3OH/HCl (10:20:0.2) followed by vigorous shaking for 5 min. The organic phase (lower phase) was separated by centrifuging for 5 min and 15 μl of the organic phase was spotted onto a silica gel thin-layer plate followed by chromatography separation for 30 minutes in CHCl₃/CH₃OH/NH₄OH/H₂O (86:76:10:14). ³²P-PI(3)P radioactivity was recorded with Typhoon 9400 Variable Imager (GE Healthcare Biosciences, Little Chalfont, UK).