Flag-Driven Immunoprecipitation of Protein Complexes

Immunoprecipitation was performed as described previously^{33 (link)}. HEK293 cells stably expressing Flag-BEX2 (1 ml) and the control cells expressing the induced pcDNA5/FRT/TO vector only (1 ml) were suspended in extraction buffer (50 mM HEPES pH 7.4, 0.3 M NaCl, 0.2% NP40) and sonicated for 5 min. The cell lysates were clarified by centrifugation at 10,000g for 30 min at 4 °C. The supernatants were filtrated through a Minisart syringe filter (Sartorius) and incubated with anti-flag antibody M2 beads (40 ml, sigma-Aldrich) for 4 h in the presence of Benzonase nuclease (10 mg/ml, Millipore) at 4 °C. After washing three times with washing buffer (0.15 M NaCl, 0.1% NP-40, 50 mM HEPES pH 7.4) and once with PBS, the binding proteins were eluted in 40 ml 0.1 M glycine buffer, pH 3.0. The eluates were neutralized with 4 ml 1 M Tris–HCl buffer pH 9.5 and suspended in SDS-PAGE (poly-acrylamide gel electrophoresis) sample buffer. The samples were boiled for 5 min and resolved by SDS-PAGE. The gel was stained using a mass silver stain kit (Wako, Osaka, Japan).