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.

Western Blotting-The cellular fractions were obtained as detailed above, and total protein concentration was determined by BCA assay (Pierce). Protein fractions (2 g) were resolved by 12% SDS-PAGE and transferred to PVDF membranes (Millipore) using a Bio-Rad semi-dry transfer apparatus. Immobilized proteins were detected with the following antibodies. Tubulin (YOL1/34) (ab6161), lamin B1 (119D5-F1) (ab8982), cell division control protein 2 homolog (Cdc2) (A17) (ab18), and nucleolin (4E2) (ab13541) were obtained from Abcam. Lamin B (101B7) (NA12) was obtained from Merck, and cyclin A (H-432) (sc-751), cyclin B1 (D-11) (sc-7393), cyclin D2 (M-20) (sc-593), and cyclin E (M-20) (sc-481) were obtained from Santa Cruz Biotechnology. HRSV proteins were detected by a goat anti-HRSV primary antibody (ab20745) from Abcam. Three HRP-conjugated secondary antibodies (A5795, A4416, and A6154) were obtained from Sigma, and one (ab6741) was from Abcam; all were detected by enhanced chemiluminescence (ECL). For the PML Western blot, 10 g of nuclear protein and 4 g of cytoplasmic protein were resolved by 15% SDS-PAGE and transferred to a PVDF membrane as described above. Immobilized proteins were detected with PML (PG-M3) (sc-966) obtained from Santa Cruz Biotechnology. The HRP-conjugated secondary antibody (A4416) was detected using SuperSignal ECL (34095) obtained from Pierce.