FIGURE 2.

Effects of PLD1 or PLD2 siRNA and catalytically inactive mPLD2 mutant on hyperoxia-induced translocation of p47^phox to cell periphery and association of p47^phox with Src and cortactin. A, HPAECs were transfected with scrambled, PLD1, or PLD2 siRNA prior to exposure to either normoxia or hyperoxia (3 h) probed with anti-p47^phox antibody, and examined by immunofluorescence microscopy using a ×60 oil objective. Exposure of cells to hyperoxia resulted in redistribution of p47^phox to cell periphery, whereas siRNA for PLD1 or PLD2 blocked p47^phox redistribution. A representative image from several experiments is shown. B, HPAECs were infected with vector control or mPLD2 K758R mutant in adenoviral construct, exposed to either normoxia or hyperoxia (3 h), and cell lysates were subjected to immunoprecipitation with anti-p47^phox antibody under non-denaturing conditions as described under “Experimental Procedures.” Immunoprecipitates were analyzed by 10% SDS-PAGE and probed with antibodies as indicated. Representative blots from three independent experiments are shown.