Time-dependent depletion of cholesterol from lipid rafts leads to the disruption of Gag-Gag, Gag-Staufen1 or Gag-IMP1 BiFC. (A) HeLa cells were co-transfected with pCMV-Rev, Gag-VN and Gag-VC. At 24 hr post-transfection the cells were stained with the Vybrant Lipid Raft Labeling Kit and treated with cholesterol disrupting drug HβCD (final concentration 30 mM). Pictures were taken at the indicated times post-HβCD treatment. (B) HeLa cells were co-transfected with pCMV-Rev and the Rev-dependent Gag  and at 24 hr post-transfection were treated with HβCD for different periods of time (as indicated, for up to 45 min). The cells were then fixed, stained for Caveolin-1 and Gag and visualized by laser scanning confocal microscopy. (C) Hela cells were co-transfected with pCMV-Rev and either Gag-VN and Staufen1-VC or Gag-VN (top panels) and IMP1-VC (lower panels). At 24 hr post-transfection lipid rafts were identified in live cells using the Vybrant Lipid Raft Labeling and treated with HβCD for up to 25 min. The BiFC signals were determined at t = 0 (not shown) and at the latest time point of t = 25 min. The size bars are equal to 10 μm.