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. 2014 Sep 26;289(47):32526–32537. doi: 10.1074/jbc.M114.606269

FIGURE 3.

FIGURE 3.

EWI-2 recruitment to EMVs is dependent on complex N-glycans. A, EWI-2 FLAG recruitment to EMVs is similar to native EWI-2. Sk-Mel-5 cells were transfected with a WT EWI-2-FLAG construct. After 48 h, culture medium was collected for EMV isolation, and TCM was isolated from cells. TCM (25 μg) and EMVs (10 μg) were analyzed for FLAG by SDS-PAGE and Western blot analysis. A higher amount of TCM was used to allow for visualization on the blot. Representative blot of three biological replicates is shown. B, EWI-2 glycans contain poly/multiantennary LacNAc structures. Sk-Mel-5 cells were transfected with wt EWI-2-FLAG. TCM was prepared from cells after 48 h. EWI-2-FLAG was immunoprecipitated from TCM samples using a mouse α-FLAG antibody. Immunoprecipitated samples were divided in two and treated either with buffer (PNGase F −) or PNGase F (PNGase F +). Treated and control samples were analyzed by SDS-PAGE and DSA lectin blot analysis. After imaging, the blot was stripped and probed with a rabbit α-FLAG antibody. Western blots shown are representative of two biological replicates. C, schematic representation of the effects of DMJ on EWI-2 glycosylation. Glycan notation follows (57). D, inhibition of complex N-glycan alters EWI-2 trafficking to EMV. Sk-Mel-5 cells were treated with DMJ (1 mm) for 48 h before collection of TCM or EMV (+ DMJ). Untreated Sk-Mel-5 cells were used as a control (− DMJ). TCM and EMV samples (+/− DMJ) were probed for EWI-2. Equal amounts of TCM samples (25 μg of protein) and of EMV (10 μg of protein) were compared by SDS-PAGE and Western blot analysis. Western blots shown are representative of two biological replicates. E, inhibition of complex N-glycan does not alter CD63 levels in EMV. TCM and EMVs from DMJ-treated cells were obtained and analyzed as in D with the exception that blots were probed with an α-CD63 antibody. F, cell surface localization of EWI-2 is not affected by DMJ treatment. Sk-Mel-5 cells were transfected with wt EWI-2-FLAG constructs and treated with either DMJ (1 mm, DMJ +) or buffer (DMJ −) for 48 h. The cells were fixed and co-stained with an α-FLAG antibody (FITC-α-FLAG, green) and DSA (biotin-DSA, Cy5-α-biotin mAb, red). Cells were imaged at multiple z-planes with fluorescence microscopy. Images were then used for deconvolution analysis with NIS Elements. A single focal plane image is shown. The scale bar is 20 μm.