Dok-1 phosphorylation in response to SDF-1α/CXCL12 is blocked by src kinase inhibitor PP2, and Dok-1 is not phosphorylated in the Lck-deficient T-cell line J.CaM1.6. (A) Jurkat cells were pretreated with src kinase inhibitor PP2 (10 μM) for 30 minutes and were left unstimulated or stimulated with SDF-1α/CXCL12 at 100 ng/mL for 5 minutes. Cell lysates were immunoprecipitated with anti–Dok-1 Ab and were immunoblotted with antiphosphotyrosine (top panel) or anti–Dok-1 Ab (bottom panel). (B) Cells were stimulated with or without SDF-1, and cell lysates were incubated with agarose-conjugated GST, GST-fyn, or GST-lck and immunoblotted with anti-Dok Ab. (C) Total cell lysates from J.CaM1.6 cells were immunoprecipitated with anti–Dok-1 Ab and then immunoblotted with antiphosphotyrosine (top panel) or anti–Dok-1 Ab (bottom panel). (D) J.CaM1.6 cells were unstimulated or stimulated with serum. Cell lysates were incubated with anti–Dok Ab and were immunoblotted with antiphosphotyrosine Ab (top panel). J.CaM1.6 cells were unstimulated or stimulated with SDF-1. Cell lysates were incubated with anti-Dok Ab and immunoblotted with antiphosphotyrosine Ab (middle panel). Cell lysates of Jurkat, J.CaM1.6, and J.CaM/Lck were immunoblotted with anti-Lck Ab (bottom panel). (E) CXCR4 expression on Jurkat and J.CaM1.6 cells. Cells were fixed with 1% paraformaldehyde PBS. Cells were incubated with anti-CXCR4 mAb or normal mouse IgG and were stained with FITC-conjugated secondary Ab and analyzed by flow cytometry. (A-E) Results in each panel are representative of at least 3 experiments each.