SDF-1–induced activation of Ral is independent of Lyn/Syk, Btk, PLC, PI3K, or Ras. DT40 cells, either wild type (WT) or deficient in Lyn/Syk, Btk, or PLCγ2 (A); DT40 cells pretreated for 30 minutes with either 20 μM LY294002 or 10 μM U73122 (B); or DT40 cells stably transfected with RasN17 (C) were stimulated for the indicated periods of time with 100 ng/mL SDF-1 and lysed, and the amount of Ral-GTP in the lysates was determined by pull-down assay (PD) using GST-RalBP-RBD fusion protein. As a control, total lysates (TLs) were immunoblotted and probed using anti-Ral, anti–P-PKB, or anti–P-MAPK (P-ERK) antibodies. (A) The right panel displays quantification of the Ral activation; the fold induction of the Ral-GTP/Ral ratio is shown as the mean (± SD) of at least 2 independent experiments. (B) DT40 cells pretreated for 30 minutes with either 20 μM LY294002, 100 nM wortmannin (WM), or 10 μM U73122 were allowed to migrate for 4 hours in the presence of the inhibitors, and in the absence (c) or presence of 100 ng/mL SDF-1 (SDF-1) in Transwells. The migration was normalized to 100% for the cells allowed to migrate in the presence of SDF-1, and shown as the mean (± SD) of triplicates. All relevant comparisons were significantly different (P < .05). (B,C) The results are representative of at least 2 independent experiments.