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

Abstract

Summary: 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
• Flow Cytometry/Mass Cytometry
• Cell-based Assays
• Molecular/Chemical Probes
• Protein expression and purification