Density Gradient Ultracentrifugation for Investigating Endocytic Recycling in Mammalian Cells
Publication in refereed journal

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其它資訊
摘要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.
出版社接受日期26.05.2021
著者Wai Wa Ray Chan, Yu Qi Zhai, Kwok-Fai Lau
期刊名稱Journal of Visualized Experiments
出版年份2021
月份6
日期30
卷號172
出版社Journal of Visualized Experiments (JoVE)
出版地USA
文章號碼e62621
國際標準期刊號1940-087X
語言美式英語
關鍵詞Density Gradient Ultracentrifugation, Endocytic Recycling

上次更新時間 2021-08-10 於 23:57