| Using 14C-acetate Pulse-chase Labeling to Study Fatty Acid and Glycerolipid Metabolism in Plant Leaves
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2021-02-05
[Abstract] Lipids metabolism is comprised of networks of reactions occurred in different subcellular compartments. Isotopic labeling is a good way to track the transformations and movements of metabolites without perturbing overall cellular metabolism. Fatty acids, the building blocks of membrane lipids and storage triacylglycerols, are synthesized in plastids. The immediate precursor for fatty acid synthesis is acetyl-CoA. Exogenous acetate is rapidly incorporated into fatty acids in leaves and isolated plastids because it can diffuse freely through cellular membranes, enter the plastid where it is rapidly metabolized to acetyl-CoA. Therefore, isotope-labeled acetate is often used as a tracer for the investigation of fatty acid synthesis and complex lipid metabolism in plants and other organisms. ...
[摘要] [摘要]脂质代谢由发生在不同亚细胞区室的反应网络组成。同位素标记是跟踪代谢物的转化和运动的好方法，而不会干扰整个细胞的新陈代谢。发TTY酸，膜脂和存储的构建块的三酰基甘油，在质体中合成的。脂肪酸合成的直接前体是乙酰辅酶A。外源乙酸盐可快速掺入叶片和分离的质体中的脂肪酸中，因为它可以通过细胞膜自由扩散，进入质体，然后迅速代谢成乙酰辅酶A。 因此，同位素标记的乙酸盐通常用作研究植物和其他生物中脂肪酸合成和复杂脂质代谢的示踪剂。同位素标记的基本原理及其最新技术进展已得到综述（Allen等，2015）。本协议描述了使用 的14 C标记的乙酸，以确定的脂肪酸合成和降解速率和跟踪的代谢甘油脂中的叶子。该方法通常被称为醋酸酯脉冲追踪标记法，已被广泛用于探查脂质代谢的各个方面（Allen等，2015），包括自噬在膜脂质更新中的作用（Fan等，2015）。，2019）和脂质与淀粉代谢途径之间的相互作用（Yu et al。，2018）。
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| The Long-lived Protein Degradation Assay: an Efficient Method for Quantitative Determination of the Autophagic Flux of Endogenous Proteins in Adherent Cell Lines
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Author:
Date:
2018-05-05
[Abstract] Autophagy is a key player in the maintenance of cellular homeostasis in eukaryotes, and numerous diseases, including cancer and neurodegenerative disorders, are associated with alterations in autophagy. The interest for studying autophagy has grown intensely in the last two decades, and so has the arsenal of methods utilised to study this highly dynamic and complex process. Changes in the expression and/or localisation of autophagy-related proteins are frequently assessed by Western blot and various microscopy techniques. Such analyses may be indicative of alterations in autophagy-related processes and informative about the specific marker being investigated. However, since these proteins are part of the autophagic machinery, and not autophagic cargo, they cannot be used to draw ...
[摘要] 自噬是维持真核生物细胞稳态的关键因素，包括癌症和神经退行性疾病在内的许多疾病都与自噬的改变有关。研究自噬的兴趣在过去的二十年里急剧增长，并且还有用于研究这种高度动态和复杂过程的方法库。通常通过Western印迹和各种显微镜技术来评估自噬相关蛋白的表达和/或定位中的变化。这样的分析可能表明自噬相关过程的改变，并且关于正在研究的特定标记物的信息。然而，由于这些蛋白质是自噬机制的一部分，而不是自噬性货物，所以它们不能用于得出关于自噬载货流量的结论。在这里，我们提供了一个协议，通过使用长寿命的蛋白质降解测定来定量评估体积自噬流量。我们的程序追踪14 C缬氨酸标记的蛋白质的降解是简单和快速的，允许并行处理相对大量的样品，并且原则上可以与任何贴壁细胞一起使用线。最重要的是，它可以通过自噬途径定量测量内源货物流量。因此，它是研究自噬活动的黄金标准之一。
【背景】脉冲追踪标记方法已用于研究蛋白质周转数十年。在此处描述的长寿命蛋白质降解（LLPD）测定中，培养细胞的蛋白质组用14 ...
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