Co-Immunoprecipitation

For native protein lysates, live cell cultures were washed with PBS and scraped. For cross-linked proteins, live cell cultures were fixed with 1% formaldehyde shaking for 10 min at room temperature. Fixation was stopped with 2.5 M Glycine by shaking for 5 min. Cells were washed with PBS and scraped. Pellets (native or cross-linked) were resuspended in lysis buffer (10 mM Tris/HCl pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 0.5% NP-40, and protease/phosphatase inhibitors) and incubated on ice for 20 minutes, and then extracts were spun down. After spinning, supernatant was collected as whole cell extract and quantified by Lowry assay. One milligram of protein was combined with beads and incubated rotating overnight at 4 °C. On the following day, protein extract was removed and beads were washed three times with wash buffer (10 mM Tris/HCl pH 7.5, 150 mM NaCl, 0.075% NP-40, and protease/phosphatase inhibitors). Beads were boiled in 2X loading buffer with DTT for 10 minutes at 95 °C. For pull down of GFP, GFP Trap (ChromoTek) beads were washed with PBS and lysis buffer before incubation with whole cell extracts. For pull down of NR2F2 Isoform 1, Dynabeads Protein A (Invitrogen) were cross-linked to NR2F2-Isoform 1 antibody (cat# 41859 Abcam, or Active Motif catalog # 61214). Beads were blocked overnight in 5% BSA at 4 °C, then washed and incubated with 5 μg antibody or AffinPure Goat Anti-Mouse IgG (Jackson) control for 2 h at 4 °C. Antibody was fixed to beads during a 30 min incubation at room temperature with 5 mM BS3 (bis(sulfosuccinimidyl)suberate) (ThermoFisher Scientific). Fixation was stopped by addition of 1 M Tris/HCl pH 7.5. Antibody-fixed beads were washed with lysis buffer before overnight incubation with whole cell extracts.