Fig. 4. PtdIns(3,4,5)P₃ activates PLD1 in vitro. (A) The effects of a range of phosphatidylinositols on the in-vitro activity of PLD1 were analyzed as described in Materials and Methods. Liposomes, reconstituted with phosphatidylethanolamine (PE), phosphatidylcholine (PC) and the indicated phosphatidylinositols, were used as substrates. Error bars indicate standard deviations. Data are representative of three independent experiments. Immunoblot in the inset indicates that the amount of PLD1 preparation used in this study were similar. (B) The dose-dependency of stimulation was checked using liposomes containing PE, PC, PtdIns and PtdIns(3,4,5)P₃. The sum of the concentrations of PtdIns and PtdIns(3,4,5)P₃ was maintained at 4.6 µM.