SARS-CoV VLP assembly and release. 293 T cells were co-transfected with SARS-CoV N, M and E expression vectors in various combinations. M-FLAG or E-FLAG indicates a FLAG tagged at the SARS-CoV M or E carboxyl terminus; HA-E denotes an HA tagged at the SARS-CoV E amino terminus. DNA (5 μg) of each plasmid was used for each transfection, with pBlueScript SK added to maintain a plasmid DNA level of 20 μg. At 48 h post-transfection, supernatants and cells were collected and prepared for protein analysis as described in Materials and Methods. Cell lysate samples (lanes 1 to 5) corresponding to 5% of total, and medium pellet samples (lanes 6 to 10) corresponding to 50% of total, were fractionated by 10% SDS-PAGE and electroblotted onto nitrocellulose filters. SARS-CoV M was probed with rabbit antiserum. SARS-CoV N and E were detected with a mouse anti-N, anti-HA, or anti-FLAG monoclonal antibody. N proteins from medium or cell samples were quantified by scanning N band densities from immunoblots. Rations of N level in media to those in cells were determined for each samples and normalized to that of samples without HA-E or E-FLAG co-expression.