FIG. 3.

Analysis of released Z-protein-containing particles. (A) Vero cell cultures were transfected with pCAGGS encoding Z and, as a control, NP. Cellular supernatants were collected, freed from cellular debris (3,000 × g, 10 min), and pelleted through a 20% sucrose cushion by UC. The UC pellet was dissolved in 100 μl of SDS-PAGE sample buffer, and transfected cells were dissolved in 500 μl of SDS-PAGE sample buffer. Twenty-microliter samples were subjected to SDS-PAGE followed by immunoblotting. NP and Z were visualized by specific immune reactions. C, cell lysate; S, pelleted material from the supernatant. (B) Protease protection assay for the identification of lipid-enveloped Z-containing particles. Aliquots of UC-pelleted Z were either treated with 0.1 μg of proteinase K alone per μl or proteinase K with 1% Triton X-100 for 30 min at 37°C or were left untreated. Samples then were analyzed by SDS-PAGE followed by immunoblotting. Masses are given in kilodaltons on left of both panels A and B.