Interactions between Gag and cellular proteins Staufen1 or IMP1 at GM1 containing lipid rafts on the plasma membrane as determined by BiFC. (A) HeLa cells were co-transfected with pCMV-Rev and Rev-dependent Gag-VN and Gag-VC plasmids. At 24 hr post-transfection lipid raft staining in live cells was performed. Images were captured using laser scanning confocal microscopy to detect the co-localization patterns of oligomerizing Gag molecules and lipid raft microdomains (indicated by CT-B that binds the pentasaccharide chain of the raft marker protein, GM1). (B) Gag-VN/Staufen1-VC BiFC signals and CT-B staining in live cells. (C) Gag-VN/IMP1-VC BiFC signals and CT-B staining in live cells. (D) HeLa cells or (E) Jurkat T cells were co-transfected with pCMV-Rev and Rev-dependent Gag-VN and Gag-VC plasmids. At 24 hr post-transfection the cells were fixed in 4% paraformaldehyde (T cells were attached to poly-D-lysine coated coverslips before fixation), permeabilized in 0.2% Triton and stained for endogenous Staufen1 and p17 to detect Gag (in Jurkat T cells only; (E), Gag is presented in blue). BiFC signals also identify Gag-Gag oligomers in fixed cells. Magnifications of cells on right show endogenous Staufen1 in Gag-Gag BiFC-negative (box 1) and positive (box 2) HeLa (D) or Jurkat T (E) cells. The size bars are equal to 10 μm.