FIG. 3.

The ORF7b protein is an integral membrane protein. 293T cells were transfected with plasmids encoding the cDNAs for GFP-Rab11, SARS-CoV GFP-N, M2 from influenza A virus, or SARS-CoV ORF7b. At 18 h posttransfection, cells were collected, lysed in hypotonic lysis buffer, and homogenized. The lysate was separated into pellet (P) and supernatant (S) fractions by centrifugation at 100,000 × g. Pellets containing cellular membranes were subsequently treated with 4 M urea, 50 mM Na₂CO₃ (pH 11.3), or 2 M KCl to disrupt peripheral membrane proteins, and the samples were centrifuged at 100,000 × g. The resultant pellet (P) and supernatant (S) were analyzed by Western blotting with rabbit anti-GFP polyclonal sera, anti-M2 MAb, or rabbit anti-ORF7b polyclonal sera.