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Density Gradient Ultracentrifugation for Investigating Endocytic Recycling in Mammalian Cells
- Source :
- Journal of Visualized Experiments.
- Publication Year :
- 2021
- Publisher :
- MyJove Corporation, 2021.
-
Abstract
- Endosomal trafficking is an essential cellular process that regulates a broad range of biological events. Proteins are internalized from the plasma membrane and then transported to the early endosomes. The internalized proteins could be transited to the lysosome for degradation or recycled back to the plasma membrane. A robust endocytic recycling pathway is required to balance the removal of membrane materials from endocytosis. Various proteins are reported to regulate the pathway, including ADP-ribosylation factor 6 (ARF6). Density gradient ultracentrifugation is a classical method for cell fractionation. After the centrifugation, organelles are sedimented at their isopycnic surface. The fractions are collected and used for other downstream applications. Described here is a protocol to obtain a recycling endosome-containing fraction from transfected mammalian cells using density gradient ultracentrifugation. The isolated fractions were subjected to standard Western blotting for analyzing their protein contents. By employing this method, we identified that the plasma membrane targeting of engulfment and cell motility 1 (ELMO1), a Ras-related C3 botulinum toxin substrate 1 (Rac1) guanine nucleotide exchange factor, is through ARF6-mediated endocytic recycling.
- Subjects :
- General Immunology and Microbiology
Chemistry
Endosome
General Chemical Engineering
General Neuroscience
Cell Membrane
Endocytic recycling
Endosomes
Endocytosis
General Biochemistry, Genetics and Molecular Biology
Protein Transport
medicine.anatomical_structure
Lysosome
Organelle
medicine
Biophysics
Animals
Centrifugation
Density gradient ultracentrifugation
Cell fractionation
Ultracentrifugation
Subjects
Details
- ISSN :
- 1940087X
- Database :
- OpenAIRE
- Journal :
- Journal of Visualized Experiments
- Accession number :
- edsair.doi.dedup.....74d92d9d75f3c7a014b9f16644f78edc