PLD2 also mediates cell migration. (A-C) Transfection controls. dHL-60 cells were transfected with pcDNA-myc-PLD2 for the length of time indicated. Cell samples were harvested, and whole lysates were taken for RNA isolation (A) for the detection of the overexpressed protein by immunoblotting with anti-PLD2 antibodies (B) (a gel loading control is presented at the bottom as the IgG heavy chain antibody), or for the subcellular detection of overexpressed protein by confocal immunofluorescence (C). (D) Cells overexpressing myc-PLD2 (or mock-transfected) were assayed for their ability to respond to chemotactic agents ENA-78, FMLP, and IL-8 (or buffer alone, Control). (E) PLD2 gene silencing. PLD2-duplex siRNA was nucleofected into HL-60 cells 1 day after differentiation induction with DMSO and challenged where appropriate with chemoattractants in Transwell. (Inset) Western blot of endogenous PLD2 and its silencing with siPLD2 RNA. (F) Effect of PA on chemokinesis. DO-PA was added at the indicated concentrations to dHL-60 cultures for 36 hours. Cells were then harvested and transferred to Transwell plates, and random cell migration was measured; 100% represents 1.1 × 10⁴ ± 0.09 cells/mL. (G) PLD2 enzymatic activity. Neutrophil suspensions were incubated with the chemoattractants for the lengths of time indicated. Total cell lysates were immunoprecipitated with anti-PLD2 antibodies, and PLD activity was assayed using immunocomplex beads. Results are mean ± SEM from 4 independent experiments conducted in duplicate. ^*P < .05 for comparison of ENA-78 versus IL-8; 100% activity represents 2152 ± 160 dpm/mg protein.