Anti dcp1a

The cells were washed with PBS and scraped after the addition of a buffer solution (160 mM Tris-HCl; pH 6.8; 4% SDS; 10% b-mercaptoethanol; 24% glycerol and 0.02% bromophenol blue) that promotes cell lysis and denatures proteins. The cell extract was collected, incubated at 95 °C for 10 min and subjected to SDS-PAGE. Then, the proteins were transferred to nitrocellulose membranes. The nitrocellulose membrane was blocked (1-h incubation with 5% milk in Tris-buffered saline—TBS), washed and incubated with the primary antibodies (anti-DDX6, MBL, Japan, PD009, 1:2500; anti-DCP1A, Santa Cruz, Dallas, TX, USA, sc-100706, 1:200 and anti-RPL30, Abcam, Cambridge, UK, ab170930). After incubation with the suitable secondary antibodies (anti-mouse IgG-alkaline phosphatase, Sigma, St. Louis, MO, USA, 1:1000 and anti-rabbit IgG-peroxidase, produced in goat, Sigma, St. Louis, MO, USA, 1:2500), the membranes were analyzed with AP buffer, BCIP e NBT (Promega, Madison, WI, USA) or with a Novex^® ECL HRP chemiluminescent kit (Invitrogen, Carlsbad, CA, USA). The signal intensity was quantified with ImageJ software [56 (link)].

A549 cells grown on Millicell EZ glass slide (Millipore) at 80% confluence were infected with WSN at an MOI of 2 for 4 to 8 h.p.i. or were transfected with 2 μg of the WSN NS1 clone. The cells were fixed in phosphate-buffered saline (PBS) containing 4% formaldehyde, permeated with 0.3% Triton X-100, blocked with 5% normal donkey serum for 1 h at 25°C, and then stained with anti-XRN1 (diluted 1:20; Santa Cruz), anti-WSN NS1 (diluted 1:100; NUS, anti-DCP1A (diluted 1:20; Santa Cruz), or anti-FLAG (diluted 1:100; Sigma) antibodies for 2 h at 37°C. Subsequently, the cells were stained with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (diluted 1:200; green, Invitrogen), goat anti-rabbit IgG (diluted 1:200; red, Invitrogen), or donkey anti-goat IgG (diluted 1:200; purple, Invitrogen) for 1 h at 25°C. The cells were washed three times with PBS and mounted in Vectashield antifade mounting medium with 4′,6′-diamidino-2-phenylindole (DAPI) (Vector Laboratories). Confocal images were obtained with a confocal laser-scanning microscope (Zeiss; LSM 700).