FIG.7.

Ability of Lnk family proteins to inhibit hematopoietic differentiation in cultured AGM cells. (A) Expression of Lnk family proteins in the AGM region. Total RNAs were extracted from E11.5 aorta and subjected to RT-PCR with specific primers for each indicated molecule. (B) The structures of Lnk, APS, and SH2-B splicing variants (SH2-B α, β, and δ) are represented schematically. (C) E11.5 AGM cells were cultured with cytokines as described above. On day 2 of culture, cells were infected with retroviruses encoding GFP or Lnk family proteins. On day 7 of culture, nonadherent cells were stained with anti-CD45 antibody, and 10⁴ cells were analyzed by flow cytometry. (D) The percentage of CD45⁺ cells in the GFP⁺ cells was determined. Error bars indicate the standard error of the mean (n = 3). (E) Flag-tagged Lnk family proteins were transfected into cultured AGM cells with a retrovirus carrying c-Kit. Cells were stimulated with SCF (100 ng/ml) for 20 min. Cell extracts were subjected to immunoprecipitation with anti-Flag antibody. Precipitates or lysates were separated by SDS-PAGE and analyzed by immunoblotting with anti-c-Kit antibody. The expression of Lnk and c-Kit was monitored.