Jafferali Mohammed Hakim, Vijayaraghavan Balaje, Figueroa Ricardo A, Crafoord Ellinor, Gudise Santhosh, Larsson Veronica J, Hallberg Einar
Department of Neurochemistry, Stockholm University, SE10691 Stockholm, Sweden.
Department of Neurochemistry, Stockholm University, SE10691 Stockholm, Sweden; Department of Biosciences and Nutrition, Karolinska Institute (Novum), SE14189 Huddinge, Sweden.
Biochim Biophys Acta. 2014 Oct;1838(10):2399-403. doi: 10.1016/j.bbamem.2014.06.008. Epub 2014 Jun 17.
Investigating interactions of proteins in the nuclear envelope (NE) using co-immunoprecipitation (Co-IP) has previously been difficult or even impossible due to their inherent resistance to extraction. We have developed a novel method, MCLIP (Membrane protein Cross-Link ImmunoPrecipitation), which takes advantage of a cell permeable crosslinker to enable effective detection and analysis of specific interactions of NE proteins in live cells using Western blot. Using MCLIP we show that, in U2OS cells, the integral inner nuclear membrane protein Samp1 interacts with Lamin B1, the LINC (Linker of nucleoskeleton and cytoskeleton) complex protein, Sun1 and the soluble small GTPase Ran. The results show that the previously detected in vitro interaction between Samp1 and Emerin also takes place in live cells. In vitro pull down experiments show, that the nucleoplasmic domains of Samp1 and Emerin can bind directly to each other. We also, show that MCLIP is suitable to coprecipitate protein interactions in different stages of the cell cycle.
由于核膜（NE）中的蛋白质具有固有的抗提取性，以前使用免疫共沉淀（Co-IP）研究其相互作用一直很困难甚至不可能。我们开发了一种新方法，即膜蛋白交联免疫沉淀（MCLIP），该方法利用一种可穿透细胞的交联剂，通过蛋白质印迹法在活细胞中有效检测和分析NE蛋白的特异性相互作用。使用MCLIP，我们发现，在U2OS细胞中，整合型内核膜蛋白Samp1与核纤层蛋白B1、LINC（核骨架与细胞骨架连接物）复合体蛋白Sun1以及可溶性小GTP酶Ran相互作用。结果表明，先前在体外检测到的Samp1与Emerin之间的相互作用在活细胞中也会发生。体外下拉实验表明，Samp1和Emerin的核质结构域可以直接相互结合。我们还表明，MCLIP适用于共沉淀细胞周期不同阶段的蛋白质相互作用。
