Mass silver stain kit

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).

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)}.

Immunoprecipitated samples were resolved on a 4–15% Mini-PROTEAN TGX gels (Bio-Rad, Hercules, CA) and stained using a Mass silver stain kit (Wako Pure Chemical Industries, Osaka, Japan). Gel slippage was reduced by 100 mM of dithiothreitol and alkylated by 100 mM idoacetamide. After washing, gels were incubated with trypsin overnight at 30 °C. Recovered peptides were desalted by Ziptip c18 (Millipore, Billerica, MA). Samples were analyzed by nanoLC-MS/MS systems (DiNa HPLC system, KYA Technologies, Tokyo, Japan; QSTAR XL, Life Technologies). Mass data acquisitions were piloted by Mascot software for matching proteins in the NCBI database.