Fig. 2. Biochemical analysis of FGF-2 fusion protein secretion. The various cell lines indicated were analyzed biochemically with regard to secretion of the reporter molecules (A). Cells were grown in the presence of doxicyclin and heparin for 48 hours at 37°C. FGF-2-GFP and GFP-FGF-2 were affinity-purified from detergent cell extracts and the medium by using heparin sepharose. 1% (cells) and 15% (medium) of the eluates were subjected to SDS-PAGE. In case of CHO_GFP cells, 1% of both cells and medium were directly subjected to SDS-PAGE (the amount of the medium loaded onto the gel had to be reduced to 1% of the total material because of the high protein concentration). Affinity-purified anti-GFP antibodies were used to detect the reporter molecules. Even after prolonged exposition, no GFP signal could be observed in lane 6. To analyze whether FGF-2-GFP is released by a specific mechanism, CHO_FGF-2-GFP cells were grown for 48 hours at 37°C in the presence of doxicyclin, 125 µg/ml heparin and 25 µM ouabain, a drug known to inhibit FGF-2 export (B). The samples were processed as described above.