title: MCLIP, an effective method to detect interactions of transmembrane proteins of the nuclear envelope in live cells.
abstract: 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.
authors.affiliation: Department of Neurochemistry, Stockholm University, SE10691 Stockholm, Sweden.
authors.forename: Mohammed Hakim
authors.initials: MH
authors.lastname: Jafferali
authors.affiliation: Department of Neurochemistry, Stockholm University, SE10691 Stockholm, Sweden.
authors.forename: Balaje
authors.initials: B
authors.lastname: Vijayaraghavan
authors.affiliation: Department of Neurochemistry, Stockholm University, SE10691 Stockholm, Sweden.
authors.forename: Ricardo A
authors.initials: RA
authors.lastname: Figueroa
authors.affiliation: Department of Neurochemistry, Stockholm University, SE10691 Stockholm, Sweden.
authors.forename: Ellinor
authors.initials: E
authors.lastname: Crafoord
authors.affiliation: Department of Neurochemistry, Stockholm University, SE10691 Stockholm, Sweden; Department of Biosciences and Nutrition, Karolinska Institute (Novum), SE14189 Huddinge, Sweden.
authors.forename: Santhosh
authors.initials: S
authors.lastname: Gudise
authors.affiliation: Department of Neurochemistry, Stockholm University, SE10691 Stockholm, Sweden.
authors.forename: Veronica J
authors.initials: VJ
authors.lastname: Larsson
authors.affiliation: Department of Neurochemistry, Stockholm University, SE10691 Stockholm, Sweden. Electronic address: einar.hallberg@neurochem.su.se.
authors.forename: Einar
authors.initials: E
authors.lastname: Hallberg
mesh_terms: D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D009685:Nuclear Envelope / Q000737:chemistry / Q000378:metabolism; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary
chemical_list: D008565:Membrane Proteins; D009687:Nuclear Proteins; C092710:emerin
keywords: CoIP; Crosslinking; Nuclear envelope; Nuclear membrane; Protein–protein interaction; Samp1