Figure 6
SHIP phosphorylation is reduced in Lyn/Hck-deficient HSCs and expression of a membrane-bound form of SHIP reverses dko phenotypes.
(A and B) SHIP phosphorylation in KSL cells and macrophages was lower in dko than WT mice, as shown by flow cytometric analysis (A) and confocal microscopy (B) using anti–p-SHIP (Tyr1020). Original magnification, ×400. (C) WT and dko BM-derived mast cells were sensitized with anti-DNP IgE and stimulated with antigen for the indicated periods. Tyrosine phosphorylation of SHIP was analyzed by immunoprecipitation followed by probing with anti-phosphotyrosine mAb (pY, upper panel) or cell lysates were directly analyzed by Western blotting using anti–p-SHIP (Tyr1020) antibody (lower panel). As a control to confirm activation of cells, blots were probed with anti–p-ERK1/2. (D and E) dko and WT KSL cells were transduced with a bicistronic retroviral vector encoding rCD2/SHIP WT, rCD2/SHIP C701A (CA), or empty vector, together with GFP, and cultured in medium containing SCF, Flt3 ligand, and IL-11. Ratios of cell numbers normalized against those of empty vector-transduced cells are plotted (D). Three days after retrovirus infection, some transduced cells (150 cells seeded) were cultured in methylcellulose containing IL-3, IL-6, SCF, and erythropoietin (E). (F) Real-time PCR analysis of mRNA expression of Stat5 target genes in dko KSL cells transduced with rCD2/SHIP WT or empty vector. (G) Dko KSL cells transduced with a bicistronic retroviral vector encoding rCD2/SHIP WT, rCD2/SHIP CA, or empty vector were i.v. injected into lethally irradiated mice. Eight weeks later, CD11b+GFP+ cells in peripheral blood were enumerated by flow cytometry. *P < 0.05.
