Isolation and Characterization of Membrane Vesicles

CPE-treated claudin-4 transfectant or Caco-2 cell cultures were centrifuged at 2, 000 × g for 3 min, and those supernatants were subjected to sequential centrifugation (30 min at 10,000 × g to collect large membrane vesicles and then 90 min at 100,000 × g to collect small membrane vesicles [49 (link), 50 (link)]). Pelleted membrane vesicles resuspended back to natural (1×) concentrations, or supernatant fractions from centrifugations, were applied to parent cells for MTT cytotoxicity testing or Western blotted with a cadherin antibody (Cell Signaling Technology, Inc.) to validate vesicle depletion from supernatants. For cadherin Western blots, equal amounts of protein were separated by SDS-PAGE on Tris gels (Bio-Rad) and transferred to a polyvinylidene difluoride (PVDF) membrane.