Gag constructs produce protein in vitro but not in mammalian cells. A) Schematic representation of the three gag expression constructs: Mtv-1 gag, HBRV gag and SM gag. CMV = human cytomegalovirus immediate early promoter; ORF = open reading frame; BGH polyA = bovine growth hormone polyadenylation site. B) HEK 293T cells and C) MEF cells were transfected with the indicated gag plasmids. Gag protein production was assayed 24 hr post-transfection by metabolic labeling with35S methionine (1 hr pulse) followed by immunoprecipitation with rabbit polyclonal anti-MMTV CA sera and an antibody to β-catenin to control for cellular equivalents. Pr77gag, Gag precursor (77 KDa) and β-catenin (98 KDa) are indicated. D) The three gag constructs were transcribed and translated in vitro in a rabbit reticulocyte lysate. A T7 promoter-driven β-galactosidase gene that produces a 110 KDa product was included as a system control (+C). E) Western immunoblot of in vitro translated Gag with MMTV anti-CA sera. In this case, the β-galactosidase reaction serves as a negative control for the antibody (-C).