Production and analysis of soluble forms of hDelta-1 and hDelta-4.

Genes encoding the extracellular domain of human Delta ligands were inserted into the expression vector pcDNA and introduced into CHO cells by electroporation. Expression of soluble proteins of Delta ligands in culture supernatants was analyzed by SDS-PAGE analysis and verified by Western blotting. (A) (i) Analysis of soluble forms of hDelta-1 and hDelta-4 by Coomassie-blue–stained SDS-PAGE gels. (ii) Western blot analysis to verify the production and purification of hDelta-1– and hDelta-4–IgG chimeras using secondary, goat anti–human IgG₁ antibodies. (B) Binding and competition studies using soluble hDelta-1– and hDelta-4–FLAG–tagged chimeras on human cord blood mononuclear and Lin⁻ cells. Results show the mean percentages ± SEM of cells detected that bind to hDelta-1– and hDelta-4–FLAG–tagged ligands. *Significant difference;P < .001. The concentration of soluble hDelta-1 or hDelta-4–FLAG was titrated, and complete saturation of cell binding was found to occur at 10 μg/mL. Prior incubation of IgG₁ chimeric Delta proteins or co-incubation of 10 mM EDTA significantly inhibited detectable binding of Delta-FLAG ligands (greater than 90% inhibition). (Inset) Binding of hDelta-1 and hDelta-4 to CB Lin⁻ cells (mean ± SEM) (n = 4). Functional binding of soluble ligands to hematopoietic cells was tested by means of FLAG tag chimera by employing secondary, fluorochrome-conjugated anti-FLAG antibody complexes as described in “Materials and methods.”