Effect of a PLD enzyme-inactive mutant. (A) dHL-60 cells were mock-transfected or transfected with WT HA-PLD1 or with a lipase-inactive PLD1 mutant (K830R). Both wild-type and lipase-inactive mutants were expressed equally at the RNA level, as assayed by QRT-PCR. (Inset) Anti-HA confocal immunofluorescence showing the pattern of intracellular expression. (B) Chemotactic response of WT- or K830R-overexpressing dHL-60 cells toward the indicated chemoattractants. The mean ± SEM from 5 independent experiments performed in duplicate are shown; 100% represents 3.0 × 104 ± 0.4 cells/mL. (C) Chemokine receptor expression levels during HL-60 cell transfection. dHL-60 cells were transfected with HA-PLD1, myc-PLD2 plasmids, or siRNAs for PLD1 or PLD2. CXCR1 and CXCR2 were detected in Western blots with specific antibodies. A gel loading control is presented at the bottom of panel C, as protein staining (GelCode Blue; Pierce Biotechnology, Rockford, IL) in the region of β-actin.
