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Fig. 6. Characterization of HASPB-N18-GFP export from CHO wild-type cells compared to CHO K3 mutant cells. (A) FACS analysis. CHO wild-type cells and CHO K3 cells were grown for 48 hours at 37°C in the presence of doxicycline (1 µg/ml). Cells were processed for FACS sorting using affinity-purified anti-GFP antibodies and APC-coupled secondary antibodies to detect exported HASPB-N18-GFP by cell surface staining. For a statistical analysis of four independent experiments, GFP-derived fluorescence and APC-derived cell surface staining of CHO wild-type cells expressing HASPB-N18-GFP was set to 100%, respectively. (B) Biochemical analysis of exported HASPB-N18-GFP in CHO wild-type cells, CHO K3 cells and various control cell lines introduced in Figs 1, 2, 3, 4 using cell surface biotinylation. The experiment was conducted exactly as described in the Materials and Methods and in the legend to Fig. 4. Input material (lane 1; 2%), streptavidin supernatant (non-biotinylated proteins, lane 2; 2%) and streptavidin-bound proteins (biotinylated proteins, lane 3; 50%) were separated on SDS gels followed by western blotting using affinity-purified anti-GFP antibodies.
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