Overexpression of HA-PLD2 or Syk enhances phosphorylation of LAT activation as well as degranulation. (A-B) RBL-2H3 cells were transfected with cDNA constructs for HA-PLD2 (PLD2 or P2), catalytically inactive PLD2K758R (PLD2m or P2m), Syk (S), or vector (–). Cells were stimulated for 7 minutes with antigen (Ag) or not stimulated (NS). LAT was immunoprecipitated from cell lysates with anti-LAT antibody, and precipitated proteins were subjected to immunoblot analysis for detection of LAT and phosphorylated LAT (pY-LAT) with anti-LAT and antiphosphotyrosine antibodies. Representative blots (A) and densitometric data (B) from 3 experiments are shown. (C) Cells were also stimulated with antigen for 7 minutes for measurement of release of the granule marker, β-hexosaminidase. Values are expressed as percent of release of β-hexosaminidase in vector-transfected cells (about 31% release) and are the mean ± SEM of values from 3 experiments. Significant increase or decrease in release: *P < .05; **P < .01.