English (United Kingdom)

https://curated-unify.zendy.io/wp-json/zendy-region/v1/featured_content/oa?rat=en

https://curated-unify.zendy.io/wp-json/zendy-region/v1/highlighted_journal/

Global: Zendy Plus annual

Presents the access of premium content as premium feature

Premium Content

Presents the keyphrase highlighting as premium feature

Keyphrase Highlighting

Presents the summarisation as premium feature

Summarisation

Insights

Presents the pdf analysis as premium feature

PDF Analysis

Presents the zaia usage as premium feature

ZAIA

Global: Zendy Tools annual

Global: Zendy Free Plan

Global: Zendy Tools monthly

Global: Zendy Plus monthly

The uptake and trafficking of cell surface receptors can be monitored by a technique called 'antibody-feeding' which uses an externally applied antibody to label the receptor on the surface of cultured, live cells. Here, we adapt the traditional antibody-feeding experiment to polarized epithelial cells (Madin-Darby Canine Kidney) grown on permeable Transwell supports. By adding two tandem extracellular Myc epitope tags to the C-terminus of the SNARE protein syntaxin 3 (Stx3), we provided a site where an antibody could bind, allowing us to perform antibody-feeding experiments on cells with distinct apical and basolateral membranes. With this procedure, we observed the endocytosis and intracellular trafficking of Stx3. Specifically, we assessed the internalization rate of Stx3 from the basolateral membrane and observed the ensuing endocytic route in both time and space using immunofluorescence microscopy on cells fixed at different time points. For cell lines that form a polarized monolayer containing distinct apical and basolateral membranes when cultured on permeable supports, e.g. , MDCK or Caco-2, this protocol can measure the rate of endocytosis and follow the subsequent trafficking of a target protein from either limiting membrane.

Tracking Endocytosis and Intracellular Trafficking of Epitope-tagged Syntaxin 3 by Antibody Feeding in Live, Polarized MDCK Cells