Autophagy is a degradative program that maintains cellular homeostasis. Autophagy defects have been described in numerous diseases. However, analysis of autophagy rates can be challenging, particularly in rare cell populations or in vivo, due to limitations in currently available tools for measuring autophagy induction. Here, we describe a method to monitor autophagy by measuring phosphorylation of the protein ATG16L1. We developed and characterized a monoclonal antibody that can detect phospho-ATG16L1 endogenously in mammalian cells. Importantly, phospho-ATG16L1 is only present on newly forming autophagosomes. Therefore, its levels are not affected by prolonged stress or late-stage autophagy blocks, which can confound autophagy analysis. Moreover, we show that ATG16L1 phosphorylation is a conserved signaling pathway activated by numerous autophagy-inducing stressors. The described antibody is suitable for western blot, immunofluorescence and immunohistochemistry, and measured phospho-ATG16L1 levels directly correspond to autophagy rates. Taken together, this phospho-antibody represents an exciting tool to study autophagy induction.

中文翻译：

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用于自噬诱导分析的抗体。

自噬是一种可维持细胞动态平衡的降解程序。自噬缺陷已在多种疾病中得到描述。然而，由于目前可用的自噬诱导测量工具的局限性，自噬速率的分析可能具有挑战性，特别是在稀有细胞群体或体内。在这里，我们描述了一种通过测量蛋白质ATG16L1的磷酸化来监测自噬的方法。我们开发并鉴定了可以在哺乳动物细胞中内源性检测磷酸-ATG16L1的单克隆抗体。重要的是，磷酸-ATG16L1仅存在于新形成的自噬体上。因此，其水平不受长时间的压力或后期自噬阻滞的影响，而后者会混淆自噬分析。而且，我们表明ATG16L1磷酸化是由许多自噬诱导应激源激活的保守的信号通路。所描述的抗体适用于蛋白质印迹，免疫荧光和免疫组化，并且测得的磷酸-ATG16L1水平直接对应于自噬速率。综上所述，这种磷酸抗体代表了一种研究自噬诱导的令人兴奋的工具。