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.

PML Localization-Coverslip-adhered A549 cell monolayers in 6-well dishes were transfected with 2 g/well FLAG-PML-I or FLAG-PML-II (40) using Lipofectamine 2000 in serum-free media according to the manufacturer's instructions. After the 2-h incubation at 37°C, the transfection mixture was removed, and the cell monolayers were infected and incubated as described above. Control wells of transfection only and media only were included to assess any background reactivity. The cells were fixed with formalin and detected with the following antibodies. A FITC-conjugated anti-HRSV-specific primary antibody (ab20391) obtained from Abcam was used to detect HRSV proteins. Both tagged PML isoforms were detected with an anti-FLAG M2 (F1804) primary antibody that was obtained from Sigma-Aldrich. Alexa Flour-conjugated secondary antibodies (A1106 and A1104) were obtained from Molecular Probes (Invitrogen). The nuclei of the above fixed cells were stained with DAPI contained within the Vectorshield (Vector Laboratories) used to mount the coverslips onto glass slides.