Kim Hyung-Seok, McKnite Autumn, Christian Jan L
Division of Hematology and Hematologic Malignancies, Department of Neurobiology, Anatomy and Internal Medicine, University of Utah School of Medicine, Salt Lake City, UT, USA.
Methods Mol Biol. 2019;1891:115-133. doi: 10.1007/978-1-4939-8904-1_9.
Bone morphogenetic proteins (Bmps) are synthesized as inactive precursors that are cleaved to generate active ligands, along with prodomain fragments that can modulate growth factor activity. Here we provide three protocols that can be used to examine the process of proteolytic activation of Bmps. The first protocol describes how to generate radiolabeled Bmp precursor proteins in Xenopus oocytes and then analyze the time course of precursor cleavage by recombinant enzymes in vitro. The second protocol details how to analyze cleavage of radiolabeled precursor proteins in Xenopus oocytes over time using pulse-chase analysis and autoradiography. This protocol can also be used to analyze folding and cleavage of radiolabeled precursor proteins at steady state. Finally, the third protocol details methods for isolating Bmp cleavage products from the blastocoele of Xenopus embryos and then analyzing them on immunoblots.
骨形态发生蛋白（Bmps）最初以无活性前体的形式合成，这些前体被切割后产生活性配体，同时还会产生可调节生长因子活性的前结构域片段。在这里，我们提供了三种可用于研究Bmps蛋白水解激活过程的方法。第一种方法描述了如何在非洲爪蟾卵母细胞中生成放射性标记的Bmp前体蛋白，然后在体外分析重组酶切割前体的时间进程。第二种方法详细介绍了如何使用脉冲追踪分析和放射自显影技术，随着时间推移分析非洲爪蟾卵母细胞中放射性标记前体蛋白的切割情况。该方法也可用于分析稳态下放射性标记前体蛋白的折叠和切割。最后，第三种方法详细介绍了从非洲爪蟾胚胎囊胚腔中分离Bmp切割产物，然后在免疫印迹上进行分析的方法。
