For indirect immunofluorescence assays, monolayers of Vero-NK cells plated on 20 mm glass coverslips in a 12-well plate were infected with the recombinant MVSchw-SARS or parental MV-Schw viruses at a multiplicity of infection (M.O.I.) of 0.05. When syncytia were clearly visible but not yet confluent (24–48 h after infection) cells were washed in d -PBS, fixed with PBS-4% paraformaldehyde for 15 min and in some instances permeabilized with PBS-0.2% triton X-100 for 10 min. Coverslips were then incubated for 60 min with anti-Ssol hyperimmune mouse ascitic fluid diluted 1/1000 in PBS-1% donkey serum (DKS). After subsequent incubation with a Cy3-conjugated anti-mouse IgG secondary antibody (Jackson Immunoresearch), the samples were mounted on slides with DAPI-containing Vectashield (Vector laboratory) and analyzed under an Axioplan 2 epifluorescence microscope (Zeiss). Pictures were acquired with an Axiocam MRm camera and processed with the Axiovision software (v 4.2, Zeiss).
For western blot assays, cytosolic cell extracts were prepared from Vero-NK cells infected with the recombinant MVSchw-SARS or parental MV-Schw viruses, essentially as described previously (Lorin et al., 2004 (link)). Proteins were separated by 8% SDS-polyacrylamide gels and transferred onto a PVDF membrane prior to immunoblotting with rabbit anti-S antibodies, as described (Callendret et al., 2007 (link)).
For western blot assays, cytosolic cell extracts were prepared from Vero-NK cells infected with the recombinant MVSchw-SARS or parental MV-Schw viruses, essentially as described previously (Lorin et al., 2004 (link)). Proteins were separated by 8% SDS-polyacrylamide gels and transferred onto a PVDF membrane prior to immunoblotting with rabbit anti-S antibodies, as described (Callendret et al., 2007 (link)).