Lyn protein depletion decreases chemotaxis of primary CD34⁺ cells and Mo7e, HL-60, and Nalm-6 cells in the presence of SDF-1. (A) Mo7e cells were incubated with control buffer or 100 ng/mL SDF-1 for 1, 3, and 30 minutes. Lyn tyrosine kinase activity was determined after immunoprecipitation from lysates of stimulated and control cells. Kinase assays were carried out on Lyn precipitates and total Lyn protein was determined by Western blotting. (B) Lyn silencing with siRNA inhibits movement of SDF-1–stimulated cells. Mo7e, HL-60, and Nalm-6 cells and CD34⁺ normal bone marrow cells were stimulated with SDF-1 (100 ng/mL) for 3 hours, 48 to 72 hours after electroporation with siRNA. We performed control Western blots for Lyn, Hck, and β-actin proteins. Additional bands, which appear in Western blots for Hck in Mo7e cells, represent Lyn (open arrow; the Lyn blot was reprobed with Hck antibodies). In HL-60, Nalm-6, and CD34⁺ cells, top panels represent Lyn, bottom panels represent β-actin. (C) Chemotaxis assays in SDF-1–stimulated cells. Values in chemotaxis assays are mean plus or minus SD (n = 4). (D) Spontaneous cell movement in the absence of SDF-1 over surfaces coated with an integrin ligand. Values are mean plus or minus SD (n = 3).