FIG. 3.

EGF induces tyrosine phosphorylation of PLD2 and its association with c-Src in A431 cells. (A) Mouse monoclonal antibody specific for PLD2 was generated as described in Materials and Methods. To analyze its specificity, PLD1 or PLD2 was transfected into COS-7 cells and the lysates were immunoblotted with anti-PLD antibody which recognizes both PLD1 and PLD2 or anti-PLD2 antibody. (B) Quiescent A431 cells were pretreated with or without a selective inhibitor of Src family tyrosine kinase, PP2 (2 μM), for 30 min and stimulated with 200 ng of EGF/ml for 20 min. Cell lysates were prepared, and 1 mg of the lysates were immunoprecipitated with anti-P-Tyr antibody or anti-c-Src antibody. Immunoprecipitated proteins were separated on SDS-PAGE gel and probed with mouse monoclonal antibody specific for PLD2. The result shown is representative of three experiments.