Fig. 5. PtdIns(3,4,5)P₃-induced activation of PLD1 promotes ERK phosphorylation. (A) 5x10⁵ HEK 293 cells depleted of endogenous PLD2, were transfected with 1 µg of wild-type PLD1 DNA. 24 hours after transfection, cells were starved for 24 hours followed by an incubation with [³H]myristic acid for 3 hours. Phosphatidylbutanol (PBt) accumulation was measured in the presence of 0.4% butanol after 2 minutes. Cells were incubated with LY 294002 (50 µM) or DMSO for 20 minutes before PDGF treatment. Error bars indicate standard deviations. Data are representative of three separate experiments. To check expression levels of PLD, the same amounts of cell lysates (20 µg) were subjected to SDS-PAGE and then immunoblotted with anti-C-terminal PLD antibody (data not shown). (B) Activation of ERK1 and ERK2 induced by the stimulation of PLD were compared. (Upper panel) The phosphorylation status of ERK1/2 was analyzed by western blotting with an anti-phosphorylated-ERK antibody. (Lower panel) Expression levels of ERK1/2. The fold-increases of ERK2 phosphorylation were calculated from the ratio of phosphorylated ERK2 to unphosphorylated ERK2 (pERK2:ERK2) determined by densitometric analysis.