or 72 h after transfection, the culture medium was harvested and pelleted by centrifugation at 2,000 x g for 20 min. Clarified supernatants were then layered through a 20% sucrose cushion (PBS) and centrifuged at 100,000 x g for 2 h at 4C in a Beckman Ti rotor (Beckman Coulter, Fullerton, CA). The pellet was resuspended on ice in 60 ?l of PBS for 1 h followed by centrifugation at 2,000 x g for 10 min at 4C. Clarified supernatants as VLPs were subject to further analysis. For the preparation of cell lysates, cells were harvested at 48 h posttransfection and lysed in 600 ?l of lysis buffer (25 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% deoxycholic acid, 1% Triton X-100, 1x protease inhibitor cocktail). The cell lysates were prepared by centrifugation at 2,000 x g at 4C for 10 min. VLPs and cell lysates were separated on a 12.5% sodium dodecyl sulfate (SDS)-polyacrylamide gel and transferred to a nitrocellulose membrane (Pierce, Rockford, IL) followed by blocking in a PBS buffer containing 0.5% Tween 20 and 5% dry milk powder. Influenza virus proteins were detected by standard Western blotting procedure coupled with the ECL system (Pierce, Rockford, IL) with specific antibodies and corresponding secondary antibodies as described above. Two pharmacological inhibitors of protein palmitoylation, 2-bromopalmitate (2BP) (Sigma-Aldrich) and cerulenin (Sigma-Aldrich), were used to address the effect of palmitoylation on VLP release by palmitoylated M1 budding. COS-1 cells were transfected with pFyn-M1 as described above. 2BP at a concentration of