Comparative_Interaction_Proteomics_GFP-tagged_MAGEC2_HEK293T

293T (Human embryonic kidney cells-ATCC CRL-3216) cells were plated at 500,000 cells per 10 cm dish and grown for 48 hours. Cells were then transfected with GFP-MAGEC1 WT or GFP-MAGEC1 L318D in a 3:1 ratio of PEI (polyethylenimine) to DNA. Cells were harvested after 48 hours and lysed in 50 mM Tris pH 7.5, 150 mM NaCl, 1% Triton X-100, protease inhibitor (cOmplete™, Mini, EDTA-free (Roche)). Cells were incubated for 25 minutes on ice and then sonicated for 5 minutes at 4°C. Cells were centrifuged at 14,000 rpm for 20 minutes at 4 °C. 500 ug lysate was then added to GFP-Trap® Agarose resin (ChromoTek) and samples were incubated rotating at 4°C for 2 hours. Following washing of the beads with 50 mM Tris pH 7.5, 150 mM NaCl, proteins were eluted by the addition of 2x Laemmli buffer and boiled for 10 minutes at 95 °C. Samples were spun down and the supernatants were sent for mass spectrometry analysis as described below. Experiments were conducted in triplicate.