p52Shc phosphorylation is Lck- and Gi-dependent. (A) Flow cytometric analysis of CXCR4 surface expression on Jurkat cells and on the Lck-deficient Jurkat T-cell variant (JCaM1). (B, left) Immunoblot analysis with anti–phospho-Shc antibodies of Shc-specific immunoprecipitates from lysates of Jurkat or JCaM1 cells, either untreated or treated for the indicated times with SDF-1α. (B, right) Immunoblot analysis with anti–ZAP-70 (top) and anti-Vav (middle) antibodies of Shc-specific immunoprecipitates from lysates of Jurkat or JCaM1 cells treated as above. Control anti-Shc blots are shown at the bottom. (C) Immunoblot analysis with anti–phospho-Shc antibodies of lysates of Jurkat cells treated as above in the presence or the absence of 20 μM PP2. A control anti-Shc blot is shown at the bottom. (D) Immunoblot analysis with anti–phospho-Shc (left) or anti–phospho-Erk (right) antibodies of lysates of Jurkat cells treated as above in the presence or the absence of 500 ng/mL PTX. (E) Immunoblot analysis with anti–phospho-Shc antibodies of lysates of Jurkat cells treated as above in the presence or the absence of 100 μM Jak2 inhibitor, AG490, or after Jak2 knock down by siRNA (> 85% reduction in Jak2-specific mRNA as assessed by laser densitometric analysis of semiquantitative RT-PCR). A representative experiment is shown in the box. Cells transfected with nonspecific siRNA (ctr siRNA) are used as Jak2 controls (Jak2 siRNA, indicated as kDa in the RT-PCR). Control blots are shown at the bottom. The migration of molecular mass markers is indicated.