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Fig. 7. Quantitative analysis of FGF-2 export in the absence or presence of aminopterin employing FACS. (A) Noninduced (grey curves) and doxicycline-induced (green curves) CHO_{FGF-2-GFP-DHFR} cells were grown in the absence (a-d) or presence (e-h) of aminopterin. Following detachment cells were processed for FACS analysis using either affinity-purified anti-GFP antibodies (a,c,e,g) or monoclonal anti-His-tag antibodies (b,d,f,h). Total GFP fluorescence (a,b,e,f), as well as cell-surface staining (c,d,g,h), were measured simultaneously using a Becton Dickinson FACSCalibur system. (B) Statistical analysis of FGF-2-GFP-DHFR export in the absence and presence of aminopterin based on expression-corrected cell-surface staining. Because aminopterin causes a significant increase of FGF-2-GFP-DHFR expression, the amount of doxicycline added to aminopterin-treated cells was titrated until an expression level was reached similar to that of cells grown in the absence of aminopterin. Under these conditions, FGF-2-GFP-DHFR export was determined on the basis of cell-surface staining. Cell-surface staining measured in the absence of aminopterin was set to 100% and compared with cell-surface staining in the presence of aminopterin (n=3).

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