Rap1 activation in human primary T cells. (A) Primary human T cells were stimulated with 100 nM SDF-1α for the indicated times in seconds, and Rap1 (Rap1^GTP) was detected by pull-down assay. Total Rap1 is shown as loading control. (B) Transient transfection of T cells with GFP-control (upper) and GFP-SPA1 vectors (lower). Twenty-four hours after transfection, cells were visualized by DIC (left) or fluorescence (right) to evaluate GFP expression and transfection efficiency. Insets show peripheral localization of GFP-SPA1 compared with GFP-control. See “Cell adhesion assay” and “Laminar shear flow assays” for image acquisition information. (C) Analysis of Rap1 activation in parental T cells (left) and GFP-control– or GFP-SPA1–expressing cells (right). Cells were stimulated with 100 nM SDF-1α for 10 seconds or 1μg/mL PMA for 5 minutes, and active Rap1 (Rap1^GTP) was determined as in panel A. Cells expressing GFP-SPA1 showed significant reduction in levels of active Rap1 compared with GFP-control-expressing cells. Total Rap1 and β-actin are shown as loading controls. (D) Rap1 activation in T cells expressing GFP-control or Rap1GAP. Cells were stimulated with PMA or SDF-1α as described in panel C, and active Rap1 (Rap1^GTP), Rap1GAP levels, and β-actin (loading control) were evaluated. Overexpression of Rap1GAP efficiently reduced levels of active Rap1. Three to 5 independent experiments were done for each panel.