Affinity Purification of GST-BEX2 Interactors

GST-BEX2 and deletion mutant constructs were inserted into the pGEX4T3 vector by PCR amplification and expressed in E. coli. Cell lysates from GST- BEX2 and deletion mutant constructs were incubated with glutathione magnetic beads (30 ml) for 1 h at 4 °C and then washed three times by washing buffer A (50 mM Tris–HCl pH 7.5, 0.3 M NaCl, 0.1% NP-40). The crude nuclear extracts were incubated with GST (control) and GST-BEX2-bound magnet beads for 12 h in the presence of Benzonase nuclease (Millipore, MA, USA) at 4 °C. After washing three times with washing buffer (0.15 M NaCl, 0.1% NP-40, 50 mM HEPES pH 7.4), GST (control) and GST-BEX2-bound proteins were eluted twice with 300 ml elution buffer (1.2 M NaCl, 50 mM HEPES pH 7.4). The eluates were concentrated and desalted with Amicon ultra-4-10k centrifugal filter units (Millipore). The eluates were resolved by SDS-PAGE and stained using a mass silver stain kit (Wako) as previously described^{33 (link)}.