Edinburgh Cancer Research UK Centre, Institute of Genetics and Cancer, University of Edinburgh, Edinburgh, UK.
Methods Mol Biol. 2022;2445:243-253. doi: 10.1007/978-1-0716-2071-7_15.
Autophagy of the endoplasmic reticulum, or ER-phagy, maintains the homeostasis of the secretory pathway. This is particularly prominent in specialized secretory cells such as the acinar cells of the exocrine pancreas. The role for such a homeostatic pathway during ageing of mammals is modelled best by in vivo genetic or pharmacologic intervention in mice. This is due to the paucity of cellular models that can maintain acinar identity outside of an animal. Here we present methods for isolation of soluble and insoluble protein fractions of ER luminal proteins from the pancreas, alongside RNA. Analysis of these macromolecules allows inference of changes in ER luminal proteostasis upon autophagy-targeted interventions. These methods will likely be more widely applicable, beyond autophagy research.
内质网自噬，或 ER 自噬，维持着分泌途径的动态平衡。这在专门的分泌细胞中尤为明显，如外分泌胰腺的腺泡细胞。这种在哺乳动物衰老过程中的稳态途径的作用，最好通过在小鼠体内进行遗传或药理学干预来建模。这是由于缺乏能够在动物体外维持腺泡特性的细胞模型所致。在这里，我们介绍了从胰腺中分离内质网腔蛋白的可溶性和不溶性蛋白部分以及 RNA 的方法。对这些大分子的分析可以推断出在自噬靶向干预后内质网腔蛋白质稳态的变化。这些方法可能具有更广泛的适用性，不仅限于自噬研究。
