Protocol for quantification of the lysosomal degradation of extracellular proteins into mammalian cells

STAR Protoc. 2021 Nov 23;2(4):100975. doi: 10.1016/j.xpro.2021.100975. eCollection 2021 Dec 17.

Abstract

Endocytic internalization of extracellular proteins plays roles in signaling, nutrient uptake, immunity, and extracellular protein quality control. However, there are few protocols for analyzing the lysosomal degradation of extracellular protein. Here, we purified secreted proteins fused with pH-sensitive GFP and acid- and protease-resistant RFP from mammalian cells and describe an internalization assay for mammalian cells. This protocol enables quantification of cellular uptake and lysosomal degradation of protein-of-interest (POI) via cell biological and biochemical analyses. For full details on the use and execution of this protocol, please refer to Itakura et al. (2020).

Keywords: Cell Biology; Cell-based Assays; Flow Cytometry/Mass Cytometry; Molecular/Chemical Probes; Protein expression and purification.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antibodies, Monoclonal
  • Endocytosis / physiology
  • Flow Cytometry / methods*
  • HEK293 Cells
  • Humans
  • Immunoblotting / methods*
  • Luminescent Proteins
  • Lysosomes* / chemistry
  • Lysosomes* / metabolism
  • Proteins* / analysis
  • Proteins* / chemistry
  • Proteins* / metabolism
  • Recombinant Proteins

Substances

  • Antibodies, Monoclonal
  • Luminescent Proteins
  • Proteins
  • Recombinant Proteins