SDF-1α induces phosphorylation of Tyr174 of Vav in human PBLs. (A) Human PBLs in suspension were treated for the indicated time points with 10 nM (100 ng/mL) SDF-1α, lysed and blotted with a polyclonal antibody against phospho-Tyr174 (pY174) Vav. The same membrane was stripped and reblotted against total Vav. A representative experiment of 4 performed is shown. (B) Kinetics of SDF-1α–induced Vav phosphorylation in human PBLs. Fold induction of pY174 Vav compared to untreated cells and corrected to total Vav is shown. Results represent the mean ± SEM of 4 independent experiments. (C) Human PBLs were allowed to spread on 50 μg/mL fibronectin or to remain in suspension and were stimulated with 10 nM SDF-1α, lysed and treated as in panel A. A representative experiment and its quantitative analysis of 4 performed is shown. (D) Dose-response of SDF-1α–induced Vav phosphorylation in human PBLs. Human PBLs were allowed to spread on 50 μg/mL fibronectin or to remain in suspension and were stimulated with the indicated dose of SDF-1α for 10 minutes, lysed, and treated as in panel A. Quantitative analysis of 4 independent experiments performed is shown. Data represent the mean ± standard deviation. (E) Dose-response of SDF-1α–induced human PBL migration. Human PBLs were allowed to migrate for 3 hours in Boyden-modified chemotaxis chambers in the presence of the indicated doses of SDF-1α (○) or RANTES (regulated upon activation normal T cell expressed and presumably secreted; •), used as a control, and migration was quantified by flow cytometry as stated in “Materials and methods.” Data represent the mean ± standard deviation of 4 independent experiments performed in triplicate.