title: Measuring receptor recycling in polarized MDCK cells.
abstract: Recycling of proteins such as channels, pumps, and receptors is critical for epithelial cell function. In this chapter we present a method to measure receptor recycling in polarized Madin-Darby canine kidney cells using an iodinated ligand. We describe a technique to iodinate transferrin (Tf), we discuss how (125)I-Tf can be used to label a cohort of endocytosed Tf receptor, and then we provide methods to measure the rate of recycling of the (125)I-Tf-receptor complex. We also show how this approach, which is easily adaptable to other proteins, can be used to simultaneously measure the normally small amount of (125)I-Tf transcytosis and degradation.
authors.affiliation: Departments of Medicine and Cell Biology, University of Pittsburgh, Pittsburgh, PA, USA.
authors.forename: Luciana
authors.initials: L
authors.lastname: Gallo
authors.affiliation: Departments of Medicine and Cell Biology, University of Pittsburgh, Pittsburgh, PA, USA.
authors.forename: Gerard
authors.initials: G
authors.lastname: Apodaca
mesh_terms: D000818:Animals; D016764:Cell Polarity; D004285:Dogs; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D061985:Madin Darby Canine Kidney Cells; D008856:Microscopy, Fluorescence; D021381:Protein Transport; D059748:Proteolysis; D011956:Receptors, Cell Surface / Q000378:metabolism*
chemical_list: D011956:Receptors, Cell Surface
keywords: Endocytosis; Iodination; MDCK cell; Receptor recycling; Transferrin; Transferrin receptor