Immunoprecipitates of biotinylated 3T3 cells to detect surface expression of Jagged (A) and Westerns of hematopoietic cell lysates to detect expression of Jagged-1 (B, C, and D). (A) Lysates were prepared from 3T3 cells infected with a retroviral vector containing sequences encoding full-length human Jagged-1 (lane 1) or parental 3T3 cells (lane 2) that had been previously biotinylated to label surface proteins. Lysates were immunoprecipitated with 9E10 antibody and resulting proteins were separated on an 8% SDS-PAGE, transferred to nitrocellulose, and probed with HRP-streptavidin and developed using ECL. (B) Protein lysates were prepared from a 3T3 cell line expressing Jagged-1 (lane 1), a parental 3T3 cell line (lane 2 ), and murine hematopoietic stromal cell lines AFT024, CFC034, 2058, 2012, and 2018 (lanes 3 through 7, respectively). (C) Protein lysates were prepared from a 3T3 cell line expressing Jagged(lane 1), primary cultured murine fetal liver stoma (lane 2), murine BM cells (lane 3), primary cultured BM stroma from C57BL/6J (lane 4), and from Balb-C mice (lane 5). (D) Protein lysates were prepared from a 3T3 cell line expressing Jagged (lane 1), Ficoll-Hypaque–separated murine BM cells (lane 2), and sorted precursors (Lin⁻Sca-1⁺c-kit⁺) (lane 3). For gels in (B), (C), and (D), proteins were separated with either an 8% SDS-PAGE (B and D) or a 6% SDS-PAGE (C), transferred to nitrocellulose, and probed with SER10, a polyclonal antibody raised against C-Serrate 1.