SDF-1α promotes association of Vav to the chemokine receptor CXCR4. (A) Human PBLs were allowed to adhere to 50 μg/mL fibronectin and treated or not with 10 nM (100 ng/mL) SDF-1α for 30 minutes, fixed, and stained for CXCR4 (red) and either pY174 Vav or total Vav (green). Yellow spots represent colocalization of CXCR4 and either pY174 Vav or total Vav. Representative fields are shown. LE, leading edge; U, uropod; arrowheads, colocalization. (B) Human PBLs in suspension were treated for the indicated time points with 10 nM (100 ng/mL) SDF-1α, lysed, and CXCR4 immunoprecipitated with a specific rabbit polyclonal antibody. A rabbit polyclonal antibody against human CD69 was employed as specificity control. Samples were resolved by SDS-PAGE and blotted against Vav and CXCR4. Fold induction represents the amount of Vav found in CXCR4 immunoprecipitates compared to that in nonstimulated cells. A representative experiment and its quantitative analysis of 3 performed is shown. (C) Human PBLs in suspension were treated for 10 minutes with 10 nM (100 ng/mL) SDF-1α, lysed, and Vav immunoprecipitated with a specific rabbit polyclonal antibody against Vav. A rabbit polyclonal antibody against human ERM proteins (90/3) was employed as specificity control. Samples were separated by SDS-PAGE and blotted against CXCR4 and Vav. Fold induction represents the amount of CXCR4 found in Vav immunoprecipitates compared to that in nonstimulated cells. A representative experiment and its quantitative analysis are shown.