MCLIP, an effective method to detect interactions of transmembrane proteins of the nuclear envelope in live cells
2014 (English)In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1838, no 10, 2399-2403 p.Article in journal (Refereed) Published
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.
Place, publisher, year, edition, pages
2014. Vol. 1838, no 10, 2399-2403 p.
Samp1, Nuclear envelope, Nuclear membrane, Crosslinking, CoIP, Protein–protein interaction
Biological Sciences Chemical Sciences Biochemistry and Molecular Biology
Research subject Neurochemistry with Molecular Neurobiology
IdentifiersURN: urn:nbn:se:su:diva-109181DOI: 10.1016/j.bbamem.2014.06.008ISI: 000340975600005OAI: oai:DiVA.org:su-109181DiVA: diva2:763439
FunderSwedish Research Council, 621-2010-448Swedish Cancer Society, 110590