Flow Cytometry Core Facility, The Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary London University, London, UK.
Methods Mol Biol. 2022;2543:155-166. doi: 10.1007/978-1-0716-2553-8_13.
Autophagy and ER stress are most often studied employing a Western blotting approach to the measurement of autophagy by LC3B upregulation and the ER stress sensor signaling proteins PERK (protein kinase R-like endoplasmic reticulum kinase), IRE1, and ATF6 which initiate protein refolding and elongation of the ER until ER homeostasis is returned. If the misfolding of proteins is increased, then ER stress is maintained, and microautophagy of the ER or specifically reticulophagy occurs. However, LC3B, PERK, protein misfolding, and changes in ER mass (reticulophagy) can also be measured in a cell cycle-dependent manner by flow cytometry and the use of antibodies, protein misfolding, and ER tracking fluorescent probes.
自噬和内质网应激通常通过 Western blot 方法进行研究，通过上调 LC3B 来测量自噬，以及内质网应激传感器信号蛋白 PERK（蛋白激酶 R 样内质网激酶）、IRE1 和 ATF6，这些蛋白启动蛋白质重折叠和内质网伸长，直到内质网恢复平衡。如果蛋白质的错误折叠增加，那么内质网应激就会持续存在，并且会发生内质网的微自噬或特异性网溶酶体自噬。然而，LC3B、PERK、蛋白质错误折叠和内质网质量的变化（网溶酶体自噬）也可以通过流式细胞术和使用抗体、蛋白质错误折叠和内质网追踪荧光探针以细胞周期依赖的方式进行测量。
