Phosphorylation of Syk is enhanced by PLD2 and by Lyn. (A-C) RBL-2H3 cells were transiently cotransfected with myc-Syk along with HA-PLD2, catalytically inactive HA-PLD2K758R cDNA plasmids, or vector (V). Transfection was performed with 25 μg plasmid per 2 × 107 cells (A) or as indicated (B-C). Cells were stimulated for 7 minutes with 25 ng/mL antigen (Ag) or not stimulated (NS). Myc-Syk was immunoprecipitated (IP) from cell lystates with antimyc antibody, and precipitated proteins were subjected to immunoblot analysis for detection of tyrosine-phosphorylated Syk (pY-Syk) and myc-Syk with antibodies against phosphotyrosine and myc, respectively. (D-E) RBL-2H3 cells were cotransfected with cDNA constructs for myc-Syk along with vector (V), flag-Lyn, HA-PLD2, or the combination of flag-Lyn and HA-PLD2. Transfection was performed with 5 μg flag-Lyn and PLD2 DNA per 2 × 107 cells (E) or as indicated (D). Cells were stimulated with antigen or not for 7 minutes, and immunoblotting was performed for detection of phosphorylated Syk and myc-Syk. Representative immunoblots from 3 experiments are shown for all 5 panels, and relative densities are indicated in italics in panel E.
