Colocalization of PLD2 with LAT and Syk on the plasma membrane. (A) RBL-2H3 cells were transiently transfected with EGFP-PLD2 plasmid and then left unstimulated (NS) or stimulated with 25 ng/mL DNP-BSA (Ag) for 7 minutes for examination by confocal microscopy. Cells were counterstained with rhodamine-labeled antibodies against LAT or Syk. Representative photomicrographs are shown. (B-C) RBL-2H3 cells transiently cotransfected with HA-PLD2 and myc-Syk cDNA plasmids or mock transfected (MOCK) were stimulated or not with antigen as in panel A. Proteins were immunoprecipitated (IP) with anti-HA (B) or antimyc (C) antibodies and then subjected to immunoblot analysis with anti-Syk or anti-HA antibodies. (D) The period of stimulation of the cotransfected RBL-2H3 cells was also varied as indicated, and immunoprecipitates obtained with anti-HA antibody were subjected to immunoblot analysis with anti-Syk or anti-HA antibodies. (E) Cells were also stimulated with antigen to calculate the rate of degranulation at the given times as determined by the release of the granule marker, β-hexosaminidase. Values are expressed as percent of cellular β-hexosaminidase that was released into the medium per minute and are the mean ± SEM of values from 3 experiments.