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LSD1 coimmunoprecipitation for mass spectrometry analysis of protein-protein interactions

Procedure

The protein content of the nuclear fraction of light and heavy samples was quantified by Bradford assay and diluted to a concentration of 2 mg/ml in IP buffer [10 mM tris HCl (pH 7.6), 150 mM NaCl, and 0.2% NP-40], supplemented with 1× protease inhibitors (Roche) and 0.5 mM PMSF. Preclearing of the lysates was achieved by incubation with protein G magnetic beads for 1 hour on a rotating wheel at 4°C. The lysates were then requantified and diluted with the IP buffer supplemented with protease inhibitors to a concentration of 1.3 mg/ml. A minor fraction $(120)$ of input was collected before adding 10 μg of anti-LSD1 antibody to each sample. All samples were then incubated on a rotating wheel at 4°C overnight. For the acquisition of the basal LSD1 interactome, 120-fold molar excess of LSD1 blocking peptide was incubated together with the antibody as negative control. This peptide competes with the bait and all its coassociated factors for the antibody binding (37). The peptide was added to the light channel in the forward experiment (Rep1 and Rep2) and to the heavy channel in the reverse experiment (Rep3). Instead, for the dynamic LSD1 interactome, the inhibitor MC_2580 was added to the heavy channel in the forward experiment and in the light one in the reverse replicate. The following day, 100 μl of Dynabeads Protein G, preequilibrated in PBS supplemented with 0.5% BSA, was added to each sample and incubated for 3 hours on a rotating wheel at 4°C. Beads were then washed three times with IP buffer and once with the washing buffer IP [10 mM tris HCl (pH 7.6), 250 mM NaCl, and 0.2% NP-40]. In the last washing step, light and heavy samples of each SILAC replicate were mixed, and the coimmunoprecipitated proteins were eluted by incubation at 95°C for 5 min with the LDS Sample Buffer (NuPAGE-Invitrogen) supplemented with 100 mM DTT. Samples were loaded on an SDS-PAGE gradient gel for subsequent protein separation and mass spectrometry analysis.

Antibody used for the preparative LSD1 coimmunoprecipitation was LSD1 (Abcam, no. 17721). The blocking peptide used as mock control was Human KDM1/LSD1 peptide (ab17763).

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