Department of Developmental and Cell Biology, University of California, Irvine, Irvine, CA 92697, USA.
STAR Protoc. 2024 Mar 15;5(1):102872. doi: 10.1016/j.xpro.2024.102872. Epub 2024 Feb 5.
Autophagy supports cell survival under different stress conditions, where ATG8-family proteins are required for autophagosome biogenesis/maturation and selective autophagy. Here, we present a protocol for studying ATG8-family protein phosphorylation using Phos-tag gel, a modified SDS-PAGE system, when the related phosphorylation site information and/or specific phospho-antibody are unavailable. We describe steps for generating GST-ATG8 proteins in bacteria, purifying S protein-Flag-SBP protein (SFB)-tagged kinasefrom cells, preparing gel, and an in vitro kinase assay. We then detail procedures for western blotting and image processing. For complete details on the use and execution of this protocol, please refer to Seo et al..
自噬在不同的应激条件下支持细胞存活，其中 ATG8 家族蛋白对于自噬体生物发生/成熟和选择性自噬是必需的。在这里，我们提出了一种使用 Phos-tag 凝胶（一种改良的 SDS-PAGE 系统）研究 ATG8 家族蛋白磷酸化的方案，当相关磷酸化位点信息和/或特定的磷酸化抗体不可用时。我们描述了在细菌中生成 GST-ATG8 蛋白、从细胞中纯化 S 蛋白-Flag-SBP 蛋白（SFB）-标记激酶、准备凝胶和体外激酶测定的步骤。然后，我们详细介绍了进行 Western blot 和图像处理的程序。有关此方案的使用和执行的完整详细信息，请参阅 Seo 等人的研究。
