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. 2016 Jan 4;35(32):4225–4234. doi: 10.1038/onc.2015.487

Figure 4.

Figure 4

(a) HEK-293 cells were transfected with a DDK-tagged MUCL1 expression vector or empty vector. After 48 h, live cells were stained with anti-DDK or anti-MUCL1 antibodies followed by staining with a fluorescently labeled secondary antibody and analyzed by FCM. Cells expressing a known DDK-tagged membrane protein (positive control) demonstrated a significant shift in fluorescence. No shift in fluorescence was detected in HEK-293 cells transfected with DDK-tagged MUCL1 by either antibody. (b) HEK-293 cells were transfected with a DDK-tagged MUCL1 expression vector or empty vector. After 48 h, DDK-tagged MUCL1 was probed for using the ThermoScientific Cell Surface Protein Isolation Kit. Cells were treated with a biotinylation reagent to label surface proteins and then collected, lysed and labeled proteins were purified using neutravidin agarose resin. The eluate contains the isolated, labeled cell surface proteins and the flow-through (FT) contains unlabeled, intracellular proteins. The experiments were repeated twice with similar results.