Membrane protein cross-link immunoprecipitation

To detect protein–protein interactions of ‘hard-to-extract’ transmembrane proteins of the nuclear envelope we used our previously described MCLIP method (Jafferali et al., 2014). U2OS Samp1a–YFP-expressing cells were synchronised either in prometaphase or in metaphase. Asynchronous cultures were used to represent interphase cells. Cells were treated with the cell permeable reversible in vivo cross-linker, dithiobis-succinimidyl-propionate (DSP, Pierce/Thermo Fisher Scientific, #22585), at a final concentration of 1 mM in cell culture medium for 15 min at room temperature (18–20°C). After crosslinking, the cells were collected by centrifugation at 800 g for 10 min and quenched with 15 mM Tris-HCl pH 7.4 for 10 min at room temperature and washed twice with ice-cold PBS. The cell pellets were re-suspended in five volumes of 7 M urea and 1% Triton X-100 containing protease inhibitor cocktail (Calbiochem, #53914), incubated on ice for 20 min and homogenised with a 23-gauge needle syringe. A part of the lysate was saved as the input and combined with an equal volume of 2× sample buffer (100 mM Tris-HCl, 4% SDS, 20% glycerol, 0.04% Bromophenol Blue, 200 mM DTT) and boiled for 10 min. The remaining lysate was diluted 8× with PBS with protease inhibitor and sonicated on ice five times for 5 s each time and cleared by centrifugation at 800 g for 5 min. The sonicates were then pre-incubated with 2% BSA-blocked protein-G–Sepharose beads (control) for 1 h at 4°C with end-over-end rotation. The pre-incubated lysate was then added to agarose beads covalently coupled to camel GFP trap antibodies (Chromotech, #gta-20) for 2 h at 4°C with end-over-end rotation. The anti-GFP beads were washed twice with wash buffer (250 mM NaCl, 10 mM Hepes, 0.5% Triton X-100, pH 7.4), the bound proteins were released, and the crosslink was reversed by addition of equal volume of 2× sample buffer containing DTT followed by incubation for 10 min at 95°C. The unbound fraction from GFP trap was concentrated by trichloroacetic acid (TCA) precipitation.