PI3K activity assay

PI3K activity was measured using a PI3K enzyme-linked immunosorbent assay kit according to the manufacturer's instructions. PI3K can convert PI(4,5)P2 to PI(3,4,5)P3. The activity of PI3K was measured using a thin layer chromatography (TLC)-based assay that used PI(4,5)P2 as a substrate. The cells were rinsed with an ice-cold buffer and harvested with an ice-cold lysis buffer. Then, PI3K was isolated from the cellular proteins by immunoprecipitation using a PI3K antibody. The immunocomplex-bound protein A-agarose beads were incubated in a reaction buffer containing PI(4,5)P2 and ATP, and the kinase reaction was stopped by centrifugation. The absorbance value of the reaction mixtures was measured at 450 nm with a microplate reader. The PI3K activity was calculated from a standard curve using various concentrations of PI(3,4,5)P3.