Colocalization of proNE and GFP-CD63 in COS cells. COS cells were transfected with plasmids carrying NE, NE plus GFP-CD63, and NE plus GFP-CD63ΔLEL (lacking the large extracellular loop) as indicated. Unpermeabilized live cells (surface) and permeabilized cells were examined by immunofluorescence microscopy after staining with anti-proNE. Surface staining of proNE is visible in all transfectants, but appears to be strongest in cells coexpressing GFP-CD63ΔLEL. In permeabilized cells expressing NE or NE plus GFP-CD63, proNE staining is concentrated in the perinuclear space and cytoplasmic organelles; the double transfectant shows colocalization of proNE and GFP. In contrast, cells coexpressing NE plus GFP-CD63ΔLEL show much lower colocalization of proNE and GFP. Images were recorded on an Eclipse TE 300 (Nikon Nordic AB, Solna, Sweden) inverted-fluorescence microscope equipped with a C4742-95 cooled CCD camera (Hamamatsu; Photonics; Norden AB, Solna, Sweden) using a plan Apochromat 60×/1.40 oil objective and ImageProPlus version 4.5.0.29 (Media Cybemetics, Bethesda, MD). Scale bar represents 10 μm.