(A) Expression of SCF mRNA by retroviral producer cells. 1MI cells transfected with the plasmid pREP8▵SCF were selected in histidinol and individual clones isolated. RNA was extracted from four independently isolated clones and analyzed by Northern blot. The blot was hybridized to an SCF cDNA probe. Lane C, pREP8▵SCF plasmid DNA digested with BamH1 and HindIII to release the SCF cDNA fragment; lanes 1 through 4, four independent clones of transfected 1MI cells. (B) Retroviral titer of fibroblast clones isolated following transfection with pREP8▵SCF. Culture supernatants were collected from four independent clones of 1MI cells following transfection with plasmid pREP8▵SCF. Twenty-five microliters (top row) or 2.5 μL (bottom row) of supernatant was spotted onto the surface of a nylon filter using a dot blot manifold and hybridized with a probe for p47^phox. Lanes 1 through 4, supernatants from four independently isolated clones; (+), retroviral genomic DNA plasmid containing p47^phox cDNA ; (−) , wild-type retroviral genomic DNA plasmid. (C) Proliferation assay on TF-cells. Biological activity of the membrane-bound growth factor on the surface of the 1MI-▵SCF cell line was assayed by ³H thymidine incorporation into quiescent TF-1 cells. sSCF, TF-1 cells stimulated with 50 ng/mL recombinant human SCF; 1MI-▵SCF, TF-1 cells cocultured for 24 hours with the 1MI-▵SCF cell line; 1MI-▵SCF + sSCF, TF-1 cells cocultured for 24 hours with the 1MI-▵SCF cell line and 50 ng/mL recombinant human SCF; 1MI-▵SCF + Transwell, TF-1 cells cocultured for 24 hours with the 1MI-▵SCF cell line, where the TF-1 cells were suspended in a Transwell. Incorporation was corrected for background values obtained with unstimulated TF-cells and 1MI-▵SCF cells, as appropriate.