Quantitative Proteomic Analysis of A549 Cells Infected with Human Respiratory Syncytial Virus

Munday, Diane C.; Emmott, Edward; Surtees, Rebecca; Lardeau, Charles-Hugues; Wu, Weining; Duprex, W. Paul; Dove, Brian K.; Barr, John N.; Hiscox, Julian A.

Enrichment of Cytoplasmic and Nuclear Proteins by Subcellular Fractionation-Cell pellets were resuspended in a cold cytoplasmic lysis buffer (20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.5 mM EDTA, 0.5% Nonidet P-40, EDTA-free Complete protease inhibitor mixture (Roche Applied Science)) and incubated for 10 min on ice. The su-pernatant containing predominantly cytoplasmic proteins was collected after a 3-min centrifugation at 2,000 ϫ g at 4°C. The remaining pellet was resuspended in radioimmune precipitation assay buffer (50 mM Tris, pH 7.5,150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, EDTA-free Complete protease inhibitor mixture (Roche Applied Science)) and incubated for 30 min at 4°C. The supernatant containing predominantly total nuclear protein was collected after a 2-min centrifugation at 13,000 ϫ g at 4°C. Both fractions were incubated for 5 min at 4°C in a sonicating water bath.