Analysis of Transforming Growth Factor ß Family Cleavage Products Secreted Into the Blastocoele of Xenopus laevis Embryos.
Analysis of Transforming Growth Factor ß Family Cleavage Products Secreted Into the Blastocoele of Xenopus laevis Embryos.
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DOI：
10.3791/62782
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发表时间：
2021-07-21
期刊：
影响因子：
--
通讯作者：
Christian JL
中科院分区：
文献类型：
--
作者：
Kim HS;Christian JL
转化生长因子超家族的两个臂分别以转化生长因子/结节或骨形态发生蛋白(BMP)配体为代表，在胚胎发育和成体动态平衡中发挥重要作用。转化生长因子家族的成员是不活跃的前体，在内质网内二聚体和折叠。前体随后被切割成配基和原结构域片段。虽然只有二聚体配体能与转化生长因子受体结合并激活下游信号，但人们越来越认识到前结构域部分对配体活性有贡献。本文描述了一种可用于鉴定转化生长因子前体蛋白激活过程中产生的切割产物的方法。首先将编码转化生长因子前体的RNA显微注射到莱维氏血吸虫胚胎中。第二天，从原肠胚囊胚中收集卵裂产物，并对其进行蛋白质印迹分析。该方案可以相对较快地完成，不需要昂贵的试剂，并且在生理条件下提供了浓缩的转化生长因子ü切割产物的来源。
The two arms of the Transforming Growth Factor ß (Tgfß) superfamily, represented by Tgfß/Nodal or Bone morphogenetic protein (Bmp) ligands, respectively, play essential roles in embryonic development and adult homeostasis. Members of the Tgfß family are made as inactive precursors that dimerize and fold within the endoplasmic reticulum. The precursor is subsequently cleaved into ligand and prodomain fragments. Although only the dimeric ligand can engage Tgfß receptors and activate downstream signaling, there is growing recognition that the prodomain moiety contributes to ligand activity. This article describes a protocol that can be used to identify cleavage products generated during activation of Tgfß precursor proteins. RNA encoding Tgfß precursors are first microinjected into X. laevis embryos. The following day, cleavage products are collected from the blastocoele of gastrula stage embryos and analyzed on Western blots. This protocol can be completed relatively quickly, does not require expensive reagents and provides a source of concentrated Tgfß cleavage products under physiologic conditions.
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