Overexpression of catalytically inactive PLD1 enhances FcϵRI-induced tyrosine phosphorylation of early signaling molecules. The indicated cell lines were sensitized, then stimulated with antigen (100 ng/mL). Lysates were immunoprecipitated with anti-FcϵRIβ Ab (A), anti-Syk Ab (B), anti-PLC-γ1 Ab (D), or anti-PLC-γ2 Ab (E), then analyzed by immunoblotting with the indicated Abs. (C) Total cell lysates were analyzed by immunoblotting with antibodies to the phosphorylated activation loop tyrosines of Syk (anti-phospho-ALSyk). The result shown is representative of 3 independent experiments, and similar results were obtained with the other cell lines. The changes in phosphorylation in the PLD-transfected cells were compared with the controls by densitometry after normalization for protein loading. The numbers are the average from the different experiments.