Cai Zhenyu, Liu Zheng-Gang
Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.
National Center for Liver Cancer, Shanghai, China.
Methods Mol Biol. 2018;1857:85-92. doi: 10.1007/978-1-4939-8754-2_8.
Programmed necrosis, also known as necroptosis, is a form of regulated necrotic cell death that is mediated by receptor-interacting protein kinases RIP1 (or RIPK1), RIP3 (or RIPK3), and the mixed lineage kinase domain-like protein, MLKL. Following the induction of programmed necrosis, MLKL is phosphorylated by RIP3 and oligomerizes and then the protein translocates to cell plasma membrane in order to execute programmed necrosis. Here, we describe a detailed protocol to detect MLKL oligomerization in necroptotic cells by Western blotting analysis under nonreducing condition. Therefore, we established the method to detect the activation of programmed necrotic pathway.
程序性坏死，也称为坏死性凋亡，是一种由受体相互作用蛋白激酶RIP1（或RIPK1）、RIP3（或RIPK3）以及混合谱系激酶结构域样蛋白MLKL介导的受调控的坏死性细胞死亡形式。在程序性坏死被诱导后，MLKL被RIP3磷酸化并寡聚化，然后该蛋白转位至细胞质膜以执行程序性坏死。在此，我们描述了一种详细的方案，用于通过非还原条件下的蛋白质印迹分析检测坏死性凋亡细胞中的MLKL寡聚化。因此，我们建立了检测程序性坏死途径激活的方法。
