Association of PLD2 with Syk is dependent on phosphorylation of Syk. (A) RBL-2H3 cells were exposed to 20 μM PP2, 120 μM piceatannol (Pi), 50 mM 1-butanol (Bu), or 50 mM tertiary butanol (tBu) for 10 minutes before stimulation with 25 ng/mL DNP-BSA (Ag) for 7 minutes or left unstimulated (NS). Endogenous Syk was immunoprecipitated with anti-Syk antibody for detection of tyrosine-phosphorylated Syk (pY-Syk) with antiphosphotyrosine antibody and Syk with anti-Syk antibody by immunoblotting. (B-C) RBL-2H3 cells transiently cotransfected with HA-PLD2 and myc-Syk plasmids were stimulated with antigen in the presence or absence of inhibitors as described for panel A. HA-PLD2 (B) and myc-Syk (C) were immunoprecipitated with agarose-conjugated antibody against HA-tag or antimyc antibody for immunoblotting and detection of HA-PLD2, myc-Syk, and their phosphorylated counterparts (pY-) with the appropriate antibody. Representative immunoblots from 3 experiments are shown.