Figure 1 | Scientific Reports

Figure 1

From: Membrane-bound Gaussia luciferase as a tool to track shedding of membrane proteins from the surface of extracellular vesicles

Figure 1

Membrane-bound Gaussia Luciferase Is Present on EVs. (A) Gaussia luciferase was fused to a transmembrane ___domain to present it on the plasma membrane and surface of EVs. SS – signal sequence, PDGFR TM - transmembrane ___domain of the platelet-derived growth factor receptor, IRES - internal ribosome entry site, GFP - green fluorescent protein, MW – predicted molecular weight of fragments of the fusion protein. (B) Schematic diagram to illustrate mbGluc labeling of cell membrane and EVs (based on the schematic presented previously8 by our group in Fig. 2A). (C) Transmission electron micrographs. EVs were isolated by UC from conditioned medium of OVCAR5 cells and immunolabeled with anti-Gluc antibodies; scale bar 100 nm. (D) Experimental flowchart of procedure to isolate EVs by UC and density gradient fractionation. (E) mbGluc activity in EVs isolated by UC and fractionated in sucrose density gradient (procedure in D); (representative of 2 experiments, bioluminescence in each fraction measured in 3 technical replicates).

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