Transactivation of the TCR by CXCR4 is required for CXCR4-dependent p52Shc phosphorylation and downstream signaling. (A) Flow cytometric analysis of CXCR4 and CD3 surface expression on Jurkat cells and the E6.157 Jurkat T-cell variant lacking surface TCR expression (TCR−). (B) Immunoblot analysis with an antibody specific for phosphorylated CD3ζ of postnuclear supernatants from Jurkat cells pretreated with either PP2, or PTX, or AG490, or after Jak2 knockdown by siRNA, and stimulated for 5 minutes with SDF-1α. (C) Anti–P-CD3ζ immunoblot of postnuclear supernatants from Jurkat and E6.157 cells treated for 5 minutes with SDF-1α. (D, left) Immunoblot analysis with anti–phospho-Shc antibodies of CD3ζ-specific immunoprecipitates from lysates of Jurkat cells activated for 1 or 5 minutes with SDF-1α. A control anti-CD3ζ blot is shown at the bottom. (D, right) Immunoblot analysis with anti–phospho-Shc antibodies of Shc-specific immunoprecipitates from lysates of Jurkat and E6.157 cells activated for 5 minutes with SDF-1α. (E) Immunoblot analysis with an antibody specific for phosphorylated Erk1/2 (left), ZAP-70 (middle), or Vav (right) of postnuclear supernatants from Jurkat and E6.157 cells treated for 5 minutes (Erk1/2) or 1 minute (ZAP-70, Vav) with SDF-1α. Control blots of the stripped filters are shown (bottom). The migration of molecular mass markers is indicated. (F) Migration of Jurkat and E6.157 cells, measured after 1-hour treatment with SDF-1α. The data are presented as relative migration, with the migration index of SDF-1α–treated Jurkat cells taken as 100% (n ≥ 3). (G) Flow cytometric analysis of surface CXCR4 on Jurkat or E6.157 cells after treatment for the indicated times with SDF-1α at 37°C. The results are plotted as percentage CXCR4 internalization (n ≥ 3). Error bars indicate SD.