SARS-CoV VLP analysis. (A-B) 293 T cells were co-transfected with E tagged with either a FLAG or HA and N, M or S expression vectors. Culture supernatants were collected 48 h post-transfection and subjected to Western immunoblotting. E proteins tagged with HA were probed with an anti-HA antibody. The lane 4 band (panel B, arrowhead) resulted from the incomplete stripping of an anti-HA probe. (C) For buoyant density gradient analysis, concentrated supernatants derived from E, E plus N, or E plus M and N transfection samples were centrifuged through a 10-40% iodixanol gradient for 16 h. Equal quantities of ten fractions were collected from top to bottom. Fraction densities were measured and SARS-CoV, E, M and N proteins analyzed by Western immunoblotting, using anti-HA, anti-M and anti-N antibodies as probes.