Fig. 1. Characterization of the cell lines CHO_FGF-2-GFP, CHO_GFP-FGF-2 and CHO_GFP. The model cell lines generated to study non-conventional export of FGF-2 were characterized with regard to genomic cDNA integration (A), western blot analysis of doxicyclin-dependent protein expression (B), analysis of doxicyclin-dependent protein expression based on fluorescence microscopy (C-H), analysis of doxicyclin-dependent protein expression based on FACS (I-K). (A) PCR analysis: CHO_FGF-2-GFP (lanes 1,4); CHO_GFP-FGF-2 (lanes 2,5); CHO_GFP (lanes 3,6). Lanes 1-3 represent PCR reactions using genomic DNA as template isolated from the cell lines indicated; lanes 4-6 represent PCR reactions using the original retroviral plasmids as template (positive controls). (B) Western blot analysis: CHO_FGF-2-GFP (lanes 1,2); CHO_GFP-FGF-2 (lanes 3,4); CHO_GFP (lanes 5,6). Total cell lysates (20 µg protein/lane) were subjected to SDS-PAGE followed by a western blot analysis using affinity-purified anti-GFP antibodies. Lanes 1, 3 and 5 correspond to cell cultures incubated in the absence of doxicyclin; lanes 2, 4 and 6 correspond to cultures incubated in the presence of doxicyclin. (C-H) Fluorescence microscopy analysis: CHO_FGF-2-GFP (C,F); CHO_GFP-FGF-2 (D,G); CHO_GFP (E,H). Panels C, D and E represent cell cultures incubated in the absence of doxicyclin; panels F, G and H represent cell cultures incubated in the presence of doxicyclin. (I-K) FACS analysis: CHO_FGF-2-GFP (I); CHO_GFP-FGF-2 (J); CHO_GFP (K). The cell populations grown in the absence of doxicyclin are shown in white, those grown in the presence of doxicyclin are shown in grey.