Combination of Tyr mutation and cytoplasmic domain truncation enhances HIV Env protein stability on cell surfaces. Protein expression was carried out by transfection of HeLa cells. At 24 h posttransfection, the cells were pulse-labeled with [35S]Met-Cys labeling mix for 1 h and then chased in complete medium for 2, 4, or 6 h, followed by surface biotinylation and immunoprecipitation as described in Materials and Methods. Protein samples were prepared and analyzed by SDS-PAGE. (A) Pulse-chase radioactive labeling analysis for expression of HIV 89.6 Env and Env/Y712S. Lanes: 1, plasmid vector pCAGGS; 2, Env, pulse only; 3, Env, 2-h chase; 4, Env, 4-h chase; 5, Env, 6-h chase; 6, Env/Y710S, pulse only; 7, Env/Y710S, 2-h chase; 8, Env/Y710S, 4-h chase; 9, Env/Y710S, 6-h chase. (B) Pulse-chase radioactive labeling analysis for expression of HIV 89.6 Env750Tr and Env750Tr/Y710S. Lanes: 1, plasmid vector pCAGGS; 2, Env750Tr, pulse only; 3, Env750Tr, 2-h chase; 4, Env750Tr, 4-h chase; 5, Env750Tr, 6-h chase; 6, Env750Tr/Y710S, pulse only; 7, Env750Tr/Y710S, 2-h chase; 8, Env750Tr/Y710S, 4-h chase; 9, Env750Tr/Y710S, 6-h chase; Pre, HIV Env precursor gp160; SU, surface subunit gp120; TM, transmembrane subunit gp41.