SOCS expression alters CXCL12-mediated Sca-1⁺c-Kit⁺Lin^–cell mobilization. (A) pEF-Flag-I/mSOCS1/ΔNGFR (SOCS1)–, pEF-Flag-I/mSOCS3/ΔNGFR (SOCS3)–, or pEF-Flag-I/ΔNGFR (vector)–transduced Lin^– cells were lysed and analyzed in Western blot with anti-Flag mAb. As control, the same membrane was developed with anti–β-actin. (B) pEF-Flag-I/mSOCS1/ΔNGFR–, pEF-Flag-I/mSOCS3/ΔNGFR–, or pEF-Flag-I/ΔNGFR–transduced Sca-1⁺c-Kit⁺Lin^– cells were allowed to migrate toward a CXCL12 gradient. The migration index was calculated as a percentage of input cells. Data represent the mean ± SD of triplicate determinations; ***P ≤ .001. (C) Flow cytometry analysis of Sca-1⁺c-Kit⁺Lin^– cells in peripheral circulation of mice reconstituted by intrafemoral injection with Lin^– cells as in panel A. Insets show the analysis of NGFR⁺ cells in the Sca-1⁺c-Kit⁺Lin^– population. Values represent the percentage of positive cells. Histograms show representative mice (n = 8). (D) Flow cytometry analysis of myeloid lineages (CD11b, Gr1) in BM from the right femur of mice reconstituted by intrafemoral injection with Lin^– cells as in panel A. Insets show analysis of NGFR⁺ cells in CD11b⁺ and Gr1⁺ cells. Values represent the percentage of positive cells. Histograms show representative mice (n = 8). (E) Cells as in panel A were stimulated in vitro with CXCL12 (50 nM, 3 minutes), lysed, and analyzed by Western blot with anti–P-JAK2 mAb. As control, the membrane was developed with anti-JAK2.