Distinct signaling pathways are activated by internal and external VEGF-KDR autocrine loops: MAPK and PI 3-K. Cells were left untreated or exposed to the internal KDR inhibitor (KDRi) or the VEGF neutralizing antibody 4.6.1 as described in “Materials and methods.” (A) Nuclear (N) and cytosolic (C) extracts from HEL cells, probed for total Erk or phosphorylated Erk 1/2. Note the reduction of Erk phosphorylation when cells are treated with the internal KDR inhibitor, while the external blocker 4.6.1 Ab has little effect. These experiments were repeated at least 3 times. (B) Nuclear (N) and cytosolic (C) protein extracts from HEL cells, treated with internal KDR inhibitor, the external blocker 4.6.1 Ab, or the PI 3-K inhibitor LY294 402, and probed for AKT and phosphorylated AKT. Note the decrease in phosphorylated AKT in cells treated with the internal KDR inhibitor, while the external Ab has little effect. These experiments are representative of 3 independent experiments. (C) FACS analysis of HL-60 cells either untreated or treated with Erk inhibitor (U0126), the PI 3-K inhibitor (LY294 402), the internal KDR inhibitor, or the external VEGF blocker Ab 4.6.1; the cells in different conditions were stained against phosphorylated Erk 1/2 or AKT, and analyzed by FACS. The percentage of positive cells was obtained by gating viable events and comparing the staining profile with an irrelevant Ab isotype control (not shown). Note the effect of the internal KDR inhibitor at reducing the levels of phosphorylated Erk in all cells tested, while the external VEGF blocker Ab 4.6.1 has little effect. Percentages indicate the proportion of cells that are stained with Ab against P-ERK or P-AKT. The results shown are representative of 3 independent experiments, and were repeated at least 3 times.