Interaction of signaling pathways. (A) Normal CD34⁺ cells were pretreated with 10 μM LY294002 or 40 μM NSC23766 for 30 minutes where indicated and subsequently stimulated with 100 ng/mL SDF-1. ERK1/2 activation was detected by Western blotting, using antibodies against phosphorylated ERK1/2 (top) or PKB (middle). The blots were reprobed with total ERK1/2 antibody, to control for equal loading (bottom). One representative experiment is shown of 4 independent experiments. (B) Normal CD34⁺ cells or HL60 cells were stimulated with 100 ng/mL SDF-1 for the indicated time, with or without pretreatment of cells with 10 μM LY294002 for 30 minutes. Activated Rac was precipitated using GST-PAK-CRIB and visualized by Western blotting with Rac antibodies (top panels). To compare the total amount of Rac protein in the samples, total lysates were analyzed by Western blotting with Rac antibodies (bottom panels). For both CD34⁺ and HL60 cells, one representative example of 3 independent experiments is shown.