<p><b>A</b>: In order to test the capacity of the purified recombinant fusion proteins to reincorporate into cell membranes, non-transfected CHO cells were incubated with the purified GPI-anchored proteins (0.9 nM) for 1 h at 37°C. As controls, two samples were treated either with identically diluted chromatography buffer (buffer) or MEM alpha medium (medium). The soluble CXCL10-mucin-Stop protein served as an additional control as it lacks a GPI anchor. All samples except the medium control contained the same percentage of chromatography buffer and detergent. Following incubation, the cells were washed and tested for the presence of the proteins on their surface by FACS staining. Dead cells were identified by 7-AAD staining and the histograms shown are gated on viable cells. The black lines indicate staining with isotype-matched control antibodies, blue lines staining with anti c-myc antibodies. Mean fluorescence intensities (MFIs) are given for each sample. This experiment was performed routinely to monitor protein quality after purification and the data shown here therefore stand representative for over 20 independent experiments. <b>B</b>: To verify the subcellular localization of the incorporated proteins, immunofluorescence microscopy was performed. Primary microvascular endothelial cells were treated with purified CXCL10-GPI, CXCL10-mucin-GPI or a buffer control diluted in culture medium, with all samples containing the same percentage of buffer to exclude artifacts. After treatment, the cells were washed, fixed with Paraformaldehyde and incorporated proteins were detected using anti c-myc primary and biotinylated secondary antibodies followed by RPE-labeled streptavidin. The figure shows fluorescence images and corresponding bright field images from a representative experiment, which was performed three times. All images within each row were acquired using the same exposure time. The black bars indicate 50 µm.</p
