CXCR4 signaling is inhibited by p52Shc mutants lacking YY239/240 or Y317. (A) Quantification of Lck autophosphorylation, as determined by in vitro kinase assays of Lck-specific immunoprecipitates from lysates of the Jurkat T-cell transfectants expressing HA-tagged p52Shc or the Shc1F/Shc2F mutants, activated for 5 minutes with SDF-1α. The results are presented for each transfectant as fold activation of SDF-1α–treated versus untreated cells (n = 2). Error bars indicate SD. (B) Immunoblot analysis with a ZAP-70 phosphospecific antibody of postnuclear supernatants of the Jurkat T-cell transfectants activated for 1 minute with 10 ng/mL SDF-1α. (C) Immunoblot analysis with antiphosphotyrosine antibodies of Vav-specific immunoprecipitates from lysates of Jurkat cells activated for 1 minute with SDF-1α. (D) Flow cytometric analysis of F-actin polymerization in response to SDF-1α treatment for 30 seconds or 1 minute in Jurkat T cells transfected with empty vector or stably expressing HA-tagged p52Shc or the Shc1F/Shc2F mutants. The data are expressed as fold increase in F-actin (detected as fluorochrome-conjugated phalloidin staining) in stimulated versus unstimulated cells (n = 3). (E-F) Immunoblot analysis with antiphosphotyrosine antibodies of LAT-specific (E) or Itk-specific (F) immunoprecipitates from lysates of the Jurkat transfectants activated for 1 minute (E) or 5 minutes (F) with SDF-1α. Control blots with the indicated antibodies are shown at the bottom. (G). Immunoblot analysis with a Erk1/2 phosphospecific antibody of postnuclear supernatants of the Jurkat T-cell transfectants activated for 5 minutes with SDF-1α. A control anti-Erk immunoblot is shown below. Vertical lines have been inserted to indicate where a gel lane was cut. These gels came from 2 different experiments. (H) Fluorimetric analysis of [Ca²⁺]_c in Jurkat T cells transfected with empty vector or stably expressing HA-tagged p52Shc or the Shc1F/Shc2F mutants. The arrow indicates the time of addition of SDF-1α. Experiment were carried out in Ca²⁺-free medium to detect Ca²⁺ release from intracellular stores. The total levels of store-associated Ca²⁺, as measured after cell solubilization by digitonin treatment in the presence of EGTA, were similar in all cell lines. Representative experiments are shown (n = 3). (I) Flow cytometric analysis of CXCR4 surface expression on Jurkat cells and the Jurkat Shc-deficient JSL1 variant. An anti-Shc immunoblot of the respective cell lysates, together with a control antiactin blot, is shown above. (J) Immunoblot analysis with ZAP-70 (left), Vav (middle), and Erk1/2 (right) phosphospecific antibodies of postnuclear supernatants of Jurkat and JSL1 cells activated for 1 minute (ZAP-70, Vav) or 5 minutes (Erk1/2) with SDF-1α. The immunoblots shown in the figure are representative of at least 3 independent experiments. (K) Fluorimetric analysis of [Ca²⁺]_c in Jurkat cells, JSL1 cells, and JSL1 cells stably transfected with a vector encoding p52/46Shc (for expression, see Figure 4). Experimental setting and data analysis were as in panel H. Representative experiments are shown (n ≥ 2).