4.5. GFP-Trap Assay and In-Gel Digestion

HEK293T cells were infected with P2-HNF4α-eGFP or empty control lentiviruses and selected with 8 µg/mL of blasticidin for 5 to 10 days until all cells in an uninfected control Petri dish were killed by the selection. In order to perform quantitative mass spectrometry, the proteome of P2-HNF4α-eGFP and empty control cells were labelled using the SILAC technique by successive amplification in heavy and light media, respectively. To perform the GFP-TRAP assay, two Petri dishes of 150 mm were used as starting material for each condition. Cells were recovered using trypsin and washed twice carefully with D-PBS 1×. Nuclear protein extracts were prepared as previously described [46]. Briefly, cells were resuspended in a hypotonic buffer (10 mM HEPES pH 7.9, 1.5 mM MgCl₂, 10 mM KCl, 0.5 mM DTT) and dounced-homogenized 25 times with a tight pestle to isolate nuclei. Nuclei were recovered by centrifugation and further purified on a sucrose gradient. Clean nuclei pellets were resuspended in RIPA 1× buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1% Nonidet P40 substitute, 0.5% Sodium deoxycholate) and sonicated five times for 10 s at 25% on a Fisher FB-120 Sonic Dismembrator with a 1/8-inch probe (Thermo Fisher Scientific). Nuclear extracts were further diluted in RIPA 1× buffer and incubated one hour at 4 °C with 40 µl of GFP-TRAP beads (Chromotek, Hauppauge, NY, USA) to immunoprecipitate the P2-HNF4α-eGFP protein and interacting partners. Beads were washed three times with RIPA 1× and two times with PBS 1× buffer. After the last wash, beads from the different conditions were pooled together before recovering the proteins by heating the beads at 95 °C for 10 min in NuPAGE LDS Sample Buffer 1× (Invitrogen-Thermo Fisher Scientific). At all steps, a protease inhibitor cocktail (Sigma-Aldrich, Oakville, Canada) was added to avoid protein degradation. The recovered proteins were then analyzed by mass spectrometry after being reduced to peptides by in-gel digestion as previously described [47].