Influence of Dok-1 overexpression on SDF-1α/CXCL12–induced chemotaxis and activation of MAPK activity. (A) CXCR4 expression on NIH3T3 cells transfected with GFP-tagged full-length Dok-1 or with empty vector. Dok or vector-transfected NIH3T3 cells were incubated with anti-CXCR4 Ab or normal mouse IgG, stained with FITC-conjugated secondary Ab, and analyzed by flow cytometry. (B) CXCR4 protein from cell lysates of Dok or vector-transfected cells as assessed after immunoblotting with anti-CXCR4 Ab. (C) Chemotaxis in response to SDF-1α/CXCL12. (Ci) NIH3T3 cells were transfected with GFP-tagged full-length Dok-1 or with empty vector. Cell migration was analyzed in 48-well chemotaxis chambers. After sorting GFP⁺ cells, 8000 cells were placed in the upper chambers. Three to 4 hours later, cells were stained using the DiffQuik kit, and the migrating cells were counted under high-power fields (× 400). Data represent the arithmetic mean ± SEM of 4 experiments. *P < .05 compared with control vector. (Cii) Baf3 cells were transfected with full-length Dok-1. Cell migration was analyzed with chemotaxis chambers. Cells (2 × 10⁵) input cells were placed in the upper chamber, and migrated cells were counted by fluorescence-activated cell sorter (FACS). Data represent the arithmetic mean ± SEM of 3 experiments. (D) MAPK activity. NIH3T3 cells were transfected with expression vector encoding Dok-1 or empty vector. Cells were stimulated with or without SDF-1α/CXCL12. Cells were fixed and stained with phospho-ERK1 Ab. MAPK activity was analyzed by flow cytometry. Data represent the arithmetic mean ± SEM of 5 experiments. *P < .05 compared with control vector. (E) Phosphorylation of ERK-1. Dok or vector-transfected NIH3T3 cells were stimulated with SDF-1/CXCL12. Cell lysates were immunoblotted with anti-phospho Erk-1 Ab, Erk-1 Ab, or Dok Ab. Data represent results of 1 of 3 similar experiments.