Wang Yingchun, Klemke Richard L
Key Laboratory of Molecular Development Biology, Insitute of Genetics and Development Bilogy, Chinese Academy of Sciences, Beijing, China.
Methods Mol Biol. 2012;757:349-65. doi: 10.1007/978-1-61779-166-6_21.
Cell migration requires actin/myosin-mediated membrane protrusion of a pseudopodium (or lamellipodium) and its attachment to the substratum. This process guides the direction of cell movement through cytoskeletal remodeling and is regulated by complex signaling networks that act spatially downstream of integrin adhesion receptors. Understanding how these regulatory networks are organized in migratory cells is important for many physiological and pathological processes, including wound healing, immune function, and cancer metastasis. Here, we describe methods for the immunoaffinity purification of phosphotyrosine proteins (pY) from pseudopodia that have been isolated from migratory cells. These methods are compatible with current mass spectrometry-based protein identification technologies and can be utilized for the large-scale identification of the pseudopodium pY proteome in various migratory cell lines, including primary and cancer cells.
细胞迁移需要肌动蛋白/肌球蛋白介导的伪足（或片状伪足）的膜突出及其与基质的附着。这个过程通过细胞骨架重塑来引导细胞运动的方向，并由在整合素粘附受体下游空间起作用的复杂信号网络调节。了解这些调节网络在迁移细胞中是如何组织的，对于许多生理和病理过程都很重要，包括伤口愈合、免疫功能和癌症转移。在这里，我们描述了从迁移细胞中分离出的伪足进行磷酸酪氨酸蛋白（pY）免疫亲和纯化的方法。这些方法与当前基于质谱的蛋白质鉴定技术兼容，可用于大规模鉴定各种迁移细胞系（包括原代细胞和癌细胞）中的伪足pY蛋白质组。
