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Quantitative Methods to Study Endocytosis and Retrograde Transport of Cargo Proteins

  • Massiullah Shafaq-Zadah
  • Estelle Dransart
  • Ludger JohannesEmail author
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Part of the Methods in Molecular Biology book series (MIMB, volume 2233)

Abstract

Endocytosis and intracellular retrograde trafficking from endosomes to the Golgi apparatus are key cellular processes. Endocytosis is directly or indirectly involved in many if not all cellular functions ranging from nutrient uptake and receptor signaling to mitosis, cell division, and migration (Scita, Di Fiore. Nature 463(7280):464–473, 2010; McMahon, Boucrot. Nat Rev Mol Cell Biol 12(8):517–533, 2011). Retrograde trafficking is emerging as a key driver for cell polarity. Robust methods are needed to quantify these processes. At the example of the bacterial Shiga toxin and the endogenous α5β1 integrin, we here describe generic methods to differentiate (1) internalized from cell surface-accessible cargo proteins and (2) endocytic cargo proteins that have reached the Golgi apparatus via the retrograde route from those that have not. The choice of antibodies or natural ligands allows to adjust these methods to virtually any chosen biological system.

Key words

Endocytosis Retrograde transport Golgi apparatus Covalent protein modification Antibodies Immunofluorescence Immunoprecipitation Western blot 

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2021

Authors and Affiliations

  • Massiullah Shafaq-Zadah
    • 1
  • Estelle Dransart
    • 1
  • Ludger Johannes
    • 1
    Email author
  1. 1.Institut Curie, PSL Research University, Cellular and Chemical Biology unit, Endocytic Trafficking and Intracellular Delivery team, U1143 INSERM, UMR3666 CNRS, 26 rue d’UlmParis Cedex 05France

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