Lack of Gag expression is not a function of timing or protein stability. A) 293T cells were transfected with the indicated gag constructs and (24, 48 or 72 hrs post-transfection) were metabolically labelled for 1 hr followed by IP with the MMTV anti-CA antibody and a control antibody to β-catenin. Pr77gag, Gag precursor. (77 KDa) and β-catenin (98 KDa). B) Steady-state levels of Gag in HEK 293T cells at 48 and 72 hrs after transfection were detected by using immunoprecipitation followed by Western blot. Triplicate transfections (a, b, c) are shown, Mo = Mock transfected cells. Pr77gag, the Gag precursor (77 KDa). C) HEK 293T cells were transfected with the indicated plasmids or mock transfected. Twenty-four hours post-transfection, the cells were subjected to a 15-min pulse followed by lysis and immunoprecipitating with the MMTV anti-CA antibody. Pr77gag, Gag precursor. D) HEK 293T cells were transfected with the indicated gag constructs or gfp and treated 24 hr later with the proteasome inhibitor MG132 for 2 hr. Gag and GFP levels were quantified by radiolabeling and immunoprecipitating with an MMTV anti-CA antibody or with an anti-GFP antibody. Lower panel shows quantification of the Gag and GFP levels in the presence or absence of the proteasome inhibitor. Data are average of three independent experiments ± SD. "+" = MG132-treated cells, "-" = DMSO-treated cells, DLU = digital light units. Pr77gag, the Gag precursor (77 KDa), β-catenin (98 KDa), and GFP (27 KDa).