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Figure 1 Construction of occludin/ZO1 probes and analysis of their efficacy and motility. (
A) In cpstFRET (cpVenus-7aa-cpCerulean), cpCerulean (cyan) is the donor and cpVenus (yellow) is the acceptor. The probes have high Förster resonance energy transfer (FRET) efficiency when donor and acceptor are parallel without external force (f: external force). The external force reduces the FRET efficiency by changing the angle of cpstFRET. (
B) Schematic representation of the occludin/ZO1 probe constructs. The cpstFRET module was inserted between amino acids 357 and 358 in the occludin probe and between amino acids 1011 and 1012 in the ZO1 probe. (
C) FRET acceptor photobleaching (FRET-AB) was used to test the efficacy of the occludin (left) and ZO1 (right) probes in HBMEC cells. FRET-AB efficiency was 17.435% for the occludin probe and 17.02% for the ZO1 probe. (