FIGURE 1.

EMV markers and complex N-glycans colocalize in discrete cell surface domains. A, fixed Sk-Mel-5 cells (4% PFA) were co-stained with DSA (biotin-DSA, Cy5-α-biotin mAb, red) and CD81 antibody (FITC-α-CD81, green). The cells were not permeabilized and staining was on the cell surface. Cells were imaged with fluorescence microscopy. The two channels were overlapped to show co-localization of CD81 and DSA. PCC was 0.876; MO was 0.941. B, Sk-Mel-5 cells were transfected with CD63-Venus construct (Venus, green), incubated for 24 h at 37 °C, fixed with PFA, and stained with DSA (biotin-DSA, Cy5-α-biotin mAb, red). The cells were imaged in multiple z-planes with fluorescence microscopy and deconvoluted. A single focal plane is shown. The two channels were overlapped to show co-localization of CD63 and DSA. The overlap coefficients were determined for CD63 positive cells only (PCC: 0.662; MO: 0.845). C, fixed Sk-Mel-5 cells (4% PFA) were stained with biotin-DSA preincubated with either chitin hydrolysate (1/5 dilution in HBSS) or HBSS followed by staining with Cy5-α-biotin mAb (red). D, fixed Sk-Mel-5 cells (4% PFA) were treated with PNGase F, endoglycosidase H (Endo H), or HBSS only (ctrl) at 37 °C for 1 h before DSA staining as before (biotin-DSA, Cy5-α-biotin mAb, red). All scale bars are 20 μm.