Oncogenic as well as dominant negative Vav impairs SDF-1α–induced human PBL polarization. (A) Schematics of the constructs employed in these assays. (B) CXCR4-expressing HEK-293 cells were transfected with the indicated Vav constructs, stimulated for 10 minutes with 10 nM SDF-1α, pull-down experiments were performed with GST-PAK-CRIB construct (which recognizes GTP-bound Rac), and SDS-PAGE–resolved samples were blotted with anti-Rac antibody. A representative experiment of 3 performed is shown. Fold induction has been corrected to the amount of Rac present in the total lysates. (C) Human PBLs were nucleofected with GFP-Vav L213Q and allowed to adhere for 30 minutes to 50 μg/mL fibronectin in the presence of 10 nM (100 ng/mL) of SDF-1α for 30 minutes, fixed, and stained for ICAM-3 (red). Representative fields are shown. (D) Human PBLs were nucleofected with GFP-Vav(Δ1-186) or GFP-Vav human PBLs were nucleofected with GFP-Vav(Δ1-186) or GFP-Vav (Δ1-186) L213Q and treated as in panel C. Representative fields are shown. (E) Quantitative analysis of the experiments shown in panels A and B. More than 300 cells per condition have been analyzed in 4 independent experiments. (F) Human PBLs were nucleofected with the indicated Vav constructs, stimulated with 1 (10 ng/mL; ▦) or 10 nM (100 ng/mL; ▪) of SDF-1α for 30 seconds or not (□), fixed, and stained for polymerized F-actin, which was subsequently measured by flow cytometry. Data represent the mean ± standard deviation of 3 experiments performed in triplicate. CH, calponin homology; Ac, acidic domain; DH (GEF), Dbl homology guanosine exchange factor; PH, pleckstrin homology; SH, Src homology; ZF, zinc finger.