p52Shc is phosphorylated in response to SDF-1α and forms a complex with Lck, ZAP-70, and Vav. (A) Immunoblot analysis with anti–phospho-Shc antibodies of Shc-specific immunoprecipitates from lysates of Jurkat T cells (left) or freshly purified human PBLs (right) either untreated or treated for the indicated times with SDF-1α. A control anti-Shc blot of the stripped filter is shown (bottom). (B) Immunoblot analysis with anti-Lck, anti–ZAP-70, and anti-Vav antibodies of Shc-specific immunoprecipitates from lysates of Jurkat T cells treated as above. A control anti-Shc blot is shown (bottom). (C) Immunoblot analysis with anti-Vav, anti–ZAP-70, and anti-Grb2 antibodies of the proteins recovered from in vitro binding assays of Jurkat T-cell lysates using a CH1-GST fusion protein. The CH1-GST protein was either unphosphorylated or phosphorylated in vitro using recombinant ZAP-70. An antiphosphotyrosine blot of the stripped filter is shown as phosphorylation control. A Coomassie staining of the input GST fusion is also shown. (D, top) Immunoblot analysis with anti-Shc antibodies of postnuclear supernatants of Jurkat T-cell lines stably transfected with either empty vector (ctr) or HA-tagged wild-type p52Shc (Shc) or p52Shc mutants lacking either YY239/240 (Shc2F) or Y317 (Shc1F). (D, bottom) Flow cytometric analysis of CXCR4 surface expression on the Jurkat transfectants. (E,F) Immunoblot analysis with anti-Lck (E) or anti–ZAP-70 and anti-Vav (F) antibodies of HA-specific immunoprecipitates from lysates of the Jurkat T-cell transfectants treated for 1 minute with SDF-1α. Control anti-Shc blots of the stripped filters are shown below. The migration of molecular mass markers is indicated.