FIGURE 4.

EWI-2 trafficking to EMV is dependent on the number of N-glycans. A, schematic of WT and mutant (N1, N12, N123) EWI-2 constructs. FLAG tag at the C terminus is shown in red. B, Sk-Mel-5 cells were transfected with wt, N1, N12, or N123 EWI-2 FLAG constructs. The cells were fixed and co-stained with α-FLAG antibody (FITC-α-FLAG, green) and ER-Tracker Red (red). Cells were imaged at varying z-planes with fluorescence microscopy. Images were deconvolved with NIS Elements, and a single focal plane from the deconvoluted images is shown for each. The scale bar is 20 μm. C, Sk-Mel-5 cells were transfected with WT, N1, N12, or N123 EWI-2 FLAG constructs. After 48 h, culture medium was collected for EMV isolation, and TCM was isolated from cells. Equal amounts of TCM samples (25 μg) and of EMV (10 μg) were analyzed for each construct by SDS-PAGE and Western blot analysis (α-FLAG antibody). A representative blot of three biological replicates is shown. D, quantification of all biological replicates of analysis shown in C. Western blots were quantified using GelQuant.NET. For each analysis, EWI-2-FLAG intensity from MV samples was normalized to the corresponding TCM samples to account for differences in expression. All data were then normalized to the WT. The graph shows the average data, and error bars represent the S.E. for the three replicates. E, TCM and EMVs from FLAG construct transfected cells were obtained and analyzed as in C with the exception that blots were probed with an α-CD63 antibody.