Modulation of FLT-1 tyrosine phosphorylation in AML cell lines. (A) Effect of various treatments on FLT-1 tyrosine phosphorylation in FLG 29.1 cells. Serum-starved FLG 29.1 cells were stimulated with BSA (250 μg/mL), VEGF₁₆₅ (100 ng/mL), TS2/16 antibody (20μg/mL), or both VEGF₁₆₅ andTS2/16, as well as with PlGF (50 ng/mL), for 30 minutes. Total cell lysates were immunoprecipitated with anti-FLT-1 antibody. Immunoprecipitates were fractionated on SDS-PAGE and subjected to WB analysis with the anti p-Tyr antibody (top). The same blot was reprobed with anti-FLT-1 antibody (bottom). Inset, Densitometric analysis of data obtained treating the cells as in panel A. The analysis was performed as described in “Materials and methods”; data are means (± SEM) of 3 separate experiments. *Statistically significant differences between samples indicated by the horizontal bars. VEGF-treated cells vs BSA-treated cells, Student t test, P = .01; TS2/16-treated cells vs BSA-treated cells, Student t test, P = .02; VEGF plus TS2/16-treated cells vs BSA-treated cells, Student t test, P = .01; PlGF-treated cells vs BSA-treated cells, Student t test, P = .023. (B) Effect of various ion channel blockers on FLT-1 tyrosine phosphorylation in FLG 29.1 cells. Serum-starved FLG 29.1 cells were stimulated with VEGF₁₆₅ andTS2/16 for 30 minutes, in the absence or presence of inhibitors of different potassium channels: Way (1 μM) or E4031 (1 μM; both specific inhibitors of hERG1 K⁺ channels); tetra-ethyl-ammonium (5 mM); a wide inhibitor of K⁺ channels, proven not to affect hERG1 channels at 5 mM concentration); C. toxin (1 μM; an inhibitor of Ca²⁺-dependent K⁺ channels that are known to be expressed in leukemia cells). Total cell lysates were treated as in panel A. Inset, Densitometric analysis of data obtained treating the cells as in panel B. The analysis was performed as described in Document S1; data are means (± SEM) of 3 separate experiments. *Statistically significant differences between samples are indicated by the horizontal bars. VEGF plus TS2/16-treated cells vs VEGF plus TS2/16 plus Way-treated cells, Student t test, P = .03 and VEGF plus TS2/16-treated cells vs VEGF plus TS2/16 plus E4031-treated cells, Student t test, P = .04. (C) Effect of VEGF plus TS2/16 treatment on FLT-1 tyrosine phosphorylation on various AML cell lines (left). Serum-starved AML cells were stimulated with BSA (250 μg/mL) or VEGF₁₆₅ (100 ng/mL) plus TS2/16 antibody (20 μg/mL) for 30 minutes. Cell lysates, IPs, and WBs were performed as described in panel A. Effect of hERG inhibitors on FLT-1 tyrosine phosphorylation of AML cell lines (right). Culture conditions, cell stimulation, IPs, and WBs were performed in the same conditions as in panel A. Inset, Densitometric analysis of FLT-1 phosphorylation in AML cell lines stimulated with BSA or VEGF plus TS2/16. The analysis was performed as described in Document S1; data are means (± SEM) of 3 separate experiments. VEGF plus TS2/16-treated cells vs BSA-treated cells, Student t test, P < .05 for all the cell lines tested. (D) Effects of VEGF₁₆₅, TS2/16, and Way on MAPK (left) and Akt phosphorylation in FLG 29.1 leukemia cells. Cell lysates were obtained from FLG 29.1 cells treated with BSA, VEGF₁₆₅, TS2/16, or VEGF plus TS2/16, the latter either in the absence or in the presence of 1 μM Way for 30 minutes. Proteins were blotted and probed with the anti p-MAPK antibody (right) or the anti phospho Akt antibodies (Document S1). Reprobing the membranes with anti ERKs antibody or anti-Akt antibody is reported in the bottom panels.