Neutrophil PLD1 enzymatic activity is increased by chemoattractants. (A) Correlation activity/chemotaxis time wise. For thick-line tracings (activity), neutrophil suspensions (5 × 10⁶ cells/mL) were incubated in the presence of the indicated chemoattractants. At the appropriate times, cells were lysed and immunoprecipitated with anti-PLD1 antibodies. Immunocomplex beads were used to assay the activity of the enzyme in vitro with [³H]-butanol and PC8 liposomes; 100% activity represents 1034 ± 76 dpm/mg protein. For thin-line tracings (chemotaxis), either IL-8, FMLP, or ENA-78 was added to the bottom wells of Transwell plates, and migrated cells were counted under the microscope at the times indicated; 100% represents 3.5 × 10⁵ ± 0.25 cells/mL. (B) Correlation activity/chemotaxis in relation to PLD inhibitors. Neutrophils were preincubated with AEBSF, ethanol, or butanol and assayed for chemotaxis (□) or were used for IP and enzyme analysis (▪). (C) Confirmation of the inhibitory effect of PLD inhibitors on chemotaxis of dHL-60 cells. Viability at the end of the assays remained greater than 90%. Results indicated in the figure are the mean ± SEM from 4 independent experiments conducted in duplicate.