PLD synergistically enhanced in STAT3 phosphorylation induced by RET/PTC tyrosine kinase. (A) ARO and TPC-1 cells were prelabeled with [3H]-myristic acid for 3 h and then treated with the various concentrations of pervanadate for 30 min as indicated, 10 min before the end of the treatment, cells were incubated with 0.3% n-butanol. Radioactivity incorporated into PtdBut was measured as described in Methods. Each bar shows mean ± SE of at least of three independent experiments. * P < 0.05, when compared with none-treated ARO cells. # P < 0.05, when compared with pervanadate-treated ARO cells. (B) ARO and TPC-1 cells were treated with the various concentrations of pervanadate for 30 min as indicated. Whole cell lysates were analyzed by SDS-PAGE and Western blotting with antibodies against phosphorylated STAT3 (Y705), RET and PLD2. To confirm equal amounts of protein were loaded, the blots were reproved using an antibody against total STAT3 and β-actin. (C) TPC-1 cells were pretreated with n-butanol for indicated concentrations or 2% t-butanol and then treated with 10 μM pervanadate for 30 min. Cell extracts were analyzed by SDS-PAGE and Western blotting with antibodies against phosphoY705-STAT3 and STAT3. (D, E) ARO cells were transiently transfected with mock vector and RET/PTC1 (D) or RET/PTC3 (E) expression plasmids. At 24 h transfection, the cells were untreated or treated with 50 μM pervanadate for 30 min, and then total cell lysates were prepared and blotted with anti-phosphoY705-STAT3, anti-STAT3 and anti-RET antibodies. The figure is representative of at least three separate experiments.