Endogenous PIK3C3 Lipid Kinase Assay

The endogenous PIK3C3 lipid kinase assay was performed according to a previous report.⁷¹ (link) Cells were lysed and immunoprecipitaed with anti-PIK3C3. Then, the immune complexes were incubated in a buffer (20 mM HEPES, 1 mM EGTA, 0.4 mM EDTA, 5 mM MgCl₂, 0.05 mM DTT, 50 mM ATP, 5 mM MnCl₂, and 50 mM DTT, pH 7.4) containing 0.2 mg/ml phosphatidylinositol (Sigma, P5766) and 5 μCi ³²P-ATP at 37 °C for 45 min. The kinase reactions were terminated by the addition of 20 μl of 8 M HCl and extracted with 160 μl chloroform:methanol (1:1). This extracted phospholipid products were separated on Silica Gel 60A (Merck, 115111). Plates were dried and followed by visualization with a Typhoon Imager (GE Healthcare Biosciences, Piscataway, NJ).