Ral mediates SDF-1–induced migration of B cells and MM cells. (A) Wild-type (WT) and stably RalN28-transfected DT40 cells were stimulated for 1 minute with 100 ng/mL SDF-1 and lysed. The amount of Ral-GTP and Rap1-GTP in the DT40 lysates was determined by pull-down assay (PD) using GST-RalBP-RBD and GST-RalGDS-RBD fusion protein and immunoblotting with anti-Ral and anti-Rap, respectively. Total lysates (TLs) were immunoblotted and probed using anti-Ral, anti–P-PKB, or anti–P-MAPK (P-ERK) antibodies. The lower (minor) band in the Ral pull-down assays of the RalN28-expressing DT40 cells represents endogenous Ral and the upper 2 (major) bands represent overexpressed HA-tagged RalN28 (*), that is, the full-length and a partial product resulting from instability of RalN28. (B,C) Wild-type (WT) and stably RalN28-transfected DT40 cells (B) or OPM-1 and NCI-H929 MM cells cotransfected with GFP and either RalN28 or RalBPΔGAP (C) were allowed to migrate for 4 hours in the absence (c) or presence of 100 ng/mL SDF-1 (SDF-1) in Transwells. The migration (of GFP-positive cells) in the presence of SDF-1 was normalized to 100%, and is shown as the mean (± SD) of triplicates. All relevant comparisons were significantly different (P < .05). The results are representative of at least 3 independent experiments. (D) OPM-1 and NCI-H929 MM cells cotransfected with GFP and either RalN28 or RalBPΔGAP were analyzed for CXCR4 expression by FACS. The results are representative of at least 3 independent experiments. (E) OPM-1 MM cells cotransfected with Renilla luciferase and either RalN28 or RalBPΔGAP were allowed to adhere to VCAM-1 for 2 minutes in the absence (c) or presence of SDF-1. The Renilla luciferase activity for the cells adhering to VCAM-1 in the presence of SDF-1 (reflecting 52% of input cells) was normalized to 100%, and shown as the mean (± SD) of 3 independent experiments. All relevant comparisons were significantly different (P < .05).