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.

Immunofluorescence-Coverslip-adhered A549 cell monolayers were infected with HRSV at an m.o.i. of 1 or 2. The plates were rocked periodically at 37°C over a 2-h incubation period before the inoculum was replaced with fresh growth media and the plate was incubated at 37°C for a further 24 h. The cells were fixed with formalin, and the following antibodies were applied. VDAC1 (20B12) (ab14734), prohibitin (II-14-10) (ab1836), TOMM20 (ab56783), and TOMM22 (ab57523) were obtained from Abcam. NADH dehydrogenase 1␤ subcomplex subunit (NDUFB) 10 (SAB1400181) and FLAG (M2) (F1804) were obtained from Sigma. Lamin B (101-B7) (NA12) was obtained from Merck, and PML (PG-M3) (sc-966) was obtained from Santa Cruz Biotechnology. Alexa Fluor-conjugated secondary antibodies (A21050, A11004, and A11061) were obtained from Molecular Probes (Invitrogen). HRSV proteins were detected by either a goat anti-HRSV primary antibody (ab20745) recognized by a FITC-conjugated secondary (ab6881) or through the use of a FITC-conjugated anti-HRSV A2-specific primary antibody (ab20391). Coverslips supporting the fixed and stained cells were mounted onto glass slides using either Vectorshield (Vector Laboratories) or Prolong Goldா antifade reagent (Invitrogen), both of which contain a DAPI counterstain to allow visualization of cell nuclei.