Abstract
Cell surface MHC class II (MHC-II) is known to internalize and can recycle back to the plasma membrane from endosomes in antigen presenting cells. We now describe a simple protocol that allows one to follow the internalization kinetics of proteins from the surface of cells. Furthermore, a simple adaptation of this assay allows one to monitor the rate of appearance of internalized proteins back to the plasma membrane. This assay allows for quantitation of trafficking of proteins on even rare subsets of cells, something that is not possible with traditional biochemical assays.
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Grant BD, Donaldson JG (2009) Pathways and mechanisms of endocytic recycling. Nat Rev Mol Cell Biol 10:597–608
Roche PA, Furuta K (2015) The ins and outs of MHC class II-mediated antigen processing and presentation. Nat Rev Immunol 15:203–216
Cella M, Engering A, Pinet V, Pieters J, Lanzavecchia A (1997) Inflammatory stimuli induce accumulation of MHC class II complexes on dendritic cells. Nature 388:782–787
Askew D, Chu RS, Krieg AM, Harding CV (2000) CpG DNA induces maturation of dendritic cells with distinct effects on nascent and recycling MHC-II antigen-processing mechanisms. J Immunol 165:6889–6895
Walseng E, Furuta K, Bosch B, Weih KA, Matsuki Y, Bakke O, Ishido S, Roche PA (2010) Ubiquitination regulates MHC class II-peptide complex retention and degradation in dendritic cells. Proc Natl Acad Sci U S A 107:20465–20470
Cho KJ, Walseng E, Ishido S, Roche PA (2015) Ubiquitination by March-I prevents MHC class II recycling and promotes MHC class II turnover in antigen-presenting cells. Proc Natl Acad Sci U S A 112:10449–10454
Walseng E, Bakke O, Roche PA (2008) Major histocompatibility complex class II-peptide complexes internalize using a clathrin- and dynamin-independent endocytosis pathway. J Biol Chem 283:14717–14727
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Walseng, E., Roche, P.A. (2019). Monitoring Protein Endocytosis and Recycling Using FACS-Based Assays. In: van Endert, P. (eds) Antigen Processing. Methods in Molecular Biology, vol 1988. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9450-2_20
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DOI: https://doi.org/10.1007/978-1-4939-9450-2_20
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Publisher Name: Humana, New York, NY
Print ISBN: 978-1-4939-9449-6
Online ISBN: 978-1-4939-9450-2
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