Expression of murine G-CSFR in parental EPRO cells and EPRO-Gr cells. Wild-type murine G-CSFR cDNA was cloned into the retroviral vector pBabe-Puro, and the construct was transduced into EML cells. EML-Gr cells then were induced to generate EPRO cells overexpressing the G-CSFR (EPRO-Gr), which were then analyzed for total G-CSFR protein expression (A) and cell surface G-CSFR expression (B). (A) Cell lysates (5 × 10³ cells/μL2 × GSB) were harvested from EPRO-Gr pools, EPRO-Gr single-cell clones, and EPRO-pBabe (vector alone). Lysates were subjected to 7% SDS-PAGE and Western analysis probing with rabbit polyclonal anti–murine G-CSF receptor. (B) Recombinant human G-CSF was biotinylated at a molar ratio of 300:1 (biotin/G-CSF) and incubated with EPRO-Gr cells at a final G-CSF concentration of 26 nM. Surface expression of G-CSFR was then determined by flow cytometry using phycoerythrin-labeled streptavidin. EPRO-pBabe cells were analyzed in tandem, and nonstreptavidin–PE-labeled cells were used as control.