Suppression of PLD2 expression with siRNA impairs tyrosine phosphorylation of Syk, LAT, and SLP as well as degranulation. (A) RBL-2H3 cells were made to transiently express HA-PLD2 and siRNAs (RNAi) directed against PLD2 or as a control green fluorescent protein (GFP). Expression of HA-PLD2 and actin was determined by immunoblotting, and expression of PLD2 mRNA was determined by RT-PCR. (B) Cells made to express the PLD2 siRNA were also stimulated with 25 ng/mL antigen (Ag) or not (NS) for 5 minutes for detection of Syk, LAT, and SLP and their tyrosine phosphorylated (pY-) counterparts by immunoblotting after immunoprecipitation. (C) Cells were also stimulated with antigen for 15 minutes to measure release of the granule marker, β-hexosaminidase. Values are expressed as percent of cellular β-hexosaminidase that was released into the medium and are the mean ± SEM of values from 3 experiments. Significant decrease in release: *P < .01. (D) Model for the dual actions of PLD2. The data suggest that PLD2 acts in a catalytically independent and dependent manner to associate directly with Syk to enhance tyrosine phosphorylation and activation of Syk and downstream targets such as LAT and SLP-76 and form phosphatidic acid (PA), which may facilitate activation of PKC and other, as yet poorly defined, mechanisms that are essential for degranulation.