Autophagy is a cellular mechanism that can generate energy for cells or clear misfolded or aggregated proteins, and upregulating this process has been proposed as a therapeutic approach for neurodegenerative diseases. Here we describe a novel set of LC3B-II and p62 time-resolved fluorescence resonance energy transfer (TR-FRET) assays that can detect changes in autophagy in the absence of exogenous labels. Lipidated LC3 is a marker of autophagosomes, while p62 is a substrate of autophagy. These assays can be employed in high-throughput screens to identify novel autophagy upregulators, and can measure autophagy changes in cultured cells or tissues after genetic or pharmacological interventions. We also demonstrate that different cells exhibit varying autophagic responses to pharmacological interventions. Overall, it is clear that a battery of readouts is required to make conclusions about changes in autophagy.

自噬是一种细胞机制，可以为细胞产生能量或清除错误折叠或聚集的蛋白质，并且已经提出将这一过程上调作为神经退行性疾病的治疗方法。在这里，我们描述了一套新颖的LC3B-II和p62时间分辨荧光共振能量转移（TR-FRET）分析方法，可以检测在没有外源标记的情况下自噬的变化。脂质LC3是自噬的标志物，而p62是自噬的底物。这些测定法可用于高通量筛选中，以鉴定新型自噬上调剂，并可在遗传或药理学干预后测量培养细胞或组织中自噬的变化。我们还证明了不同的细胞对药理学干预表现出不同的自噬反应。全面的，