Rem in trans along with RmRE rescues Gag expression from an artificial intron. A) Schematic representation of the gag constructs that contain the RmRE. First the RmRE was introduced in the 3'UTR of pcDNA3.1 gag construct, downstream of the gag stop codon, but upstream of the polyA site (CRmRE). In a second construct gag was inserted between the second splice donor and splice acceptor from the MMTV infectious molecular clone, and upstream of the RmRE and the viral 3'LTR (CssRmRE). Mtv-1, HBRV, and SM gags from the CRmRE B) and CssRmRE C) were expressed in HEK 293T cells in the presence or absence of Rem. Lanes indicate 1, GFP; 2, Rem; 3, GFP-Rem; 4, GFP-RemSP; +, GFP-Rem; -, GFP co-transfections. Gag expression was assayed 24 hrs post-transfection by radiolabeling and immunoprecipitating with MMTV anti-CA and anti-β-catenin antibodies. Rem expression in the same samples CRmRE D) and CssRmRE E) is shown by Western blot with a rabbit polyclonal anti-GFP antibody. Pr77gag, Gag precursor (77 KDa), β-catenin (98 KDa), GFP-RemSP (40 KDa) and GFP (27 KDa). Gag levels from CRmRE F) and CssRmRE G) were quantified relative to β-catenin ± SD in the presence and absence of different Rem constructs. Lanes indicate 1, GFP; 2, Rem; 3, GFP-Rem; 4, GFP-RemSP; +, GFP-Rem; -, GFP. Data are average of three independent transfections.