Association of PLD2 and Syk, although dependent on initial phosphorylation of Syk, results in additional phosphorylation and the activation of Syk. (A) Immunoprecipitated (IP) Syk from RBL-2H3 cells was incubated initially in the presence or absence of 20 μM ATP before addition of lysates of cells that had been transfected with HA-PLD2 cDNA or not (MOCK). The precipitated proteins were subjected to immunoblot analysis for detection of HA-PLD2 and phosphorylated Syk (pY-Syk) as described in “Materials and methods.” (B) Free recombinant Syk was phosphorylated with or without ATP and HA-PLD2 that had isolated by the Catch and Release system as described in “Materials and methods.” An equal amount of BSA was used as the negative control for HA-PLD2. The mixtures were subjected to immunoblot analysis for detection of phosphorylated Syk, HA-PLD2, and Syk. Representative immunoblots from 3 experiments are shown.(C) Free recombinant Syk was assayed for kinase activity in the absence or presence of the indicated amounts of HA-PLD2 as described for panel B by use of an in vitro kinase assay kit. An equal amount of BSA (B) was used as the negative control for each HA-PLD2 sample. Values are the mean ± SEM of 3 separate experiments.