Rem, the RmRE and natural splice donor and acceptor sequences rescue Gag expression. A) Schematic representation of the MMTV infectious molecular clone (pHyb-Mtv) and the two subviral clones created (LTRgag and LTR+SA). LTR, long terminal repeat; SD, splice donor; SA, splice acceptor, RmRE, Rem response element. gag, pro, pol, env, sag are the viral genes. Expression of Gag from pLTRgag B) or pLTR+SA C) was assayed in 293T cells by pulse labeling and immunoprecipitating with an MMTV anti-CA antibody in the presence or absence of GFP-Rem. Transfection of pHyb-Mtv served as a positive control. '-' lanes were transfected with GFP to equilibrate the plasmid load. β-catenin was used as a cellular control. Expression of GFP and GFP-Rem was visualized by immunoblotting with an anti-GFP antibody in pLTRgag D) or pLTR+SA E) transfected cells. Pr77gag, the Gag precursor (77 KDa), β-catenin (98 KDa), GFP-Rem (66 KDa), GFP-RemSP (40 KDa) and GFP (27 KDa). Gag levels from the LTRgag F) and LTR+SA G) were quantified relative to β-catenin. Data are average of three independent transfections ± SD.